Science topics: Biological ScienceGeneral BiochemistryBiochemical MethodsCytologyCytological Techniquescytogenetic analysisFluorescence In Situ HybridizationScience methodFluorescence In Situ Hybridization - Science methodFluorescence In Situ Hybridization is a cytogenetic technique that is used to detect and localize the presence or absence of specific DNA sequences on chromosomes. FISH uses fluorescent probes that bind to only those parts of the chromosome with which they show a high degree of sequence complementarity. Fluorescence microscopy can be used to find out where the fluorescent probe is bound to the chromosomes.Questions (204)Publications (42,175)Questions related to Fluorescence In Situ Hybridization123 Emily Mcnallyasked a question related to Fluorescence In Situ HybridizationHow do you fluorescently label mRNA for microscopy?Question3 answersJun 11, 2021Hi all,I would like to fluorescently label mRNA that we will be delivering to MoDCs. I m going to perform microscopy (Axios Imager D2 if that s relevant) to see localization of the specific mRNA within the cells. This mRNA is from a vendor so I can t transcribe it myself. Any thoughts?Relevant answerVolkan ErginJun 11, 2021AnswerIt doesn t sound like a good idea to use a fluorophore-labeled mRNA for imaging purposes inside cells. Instead, you would try a hybridization probe against your mRNA with their related amplifiers to make it visible. Otherwise, labeled single mRNA molecules will likely be stay below the signal detection threshold under a microscope even with 63 or 100X objectives. I have been using the RNAscope system to visualize endogenous mRNAs at single-cell resolution, which I think what you need. In your case, you may request your vendor to use synonymous codons for certain amino acids if need in order to avoid cross-labeling endogenous mRNA. But sample processing is quite tedious, though could be very accurate.View6 Recommendations Asif Jokhioasked a question related to Fluorescence In Situ HybridizationWhere we can purchase DNA fish probe for Fluorescence in situ hybridization ? Question4 answersJul 31, 2018 we are working on FISH fluorescence in situ hybridization ,so we want to purchase DNA FISH probes sequence give any suggestion where i can purchase DNA FISH PROBES OR give any international vendors email or other. thanksRelevant answerYan ZhangApr 27, 2021AnswerHi Matthew,How is your experience with AUM BIoTech? Does customer need to provide probe sequences? ThanksView0 Recommendations Jorge Enrique Vazquez Buchelliasked a question related to Fluorescence In Situ HybridizationWhy im having low probe specificity on Fluorescent in situ hybridization in bacterial suspension?Question3 answersOct 16, 2019 I am using specific probes for detecting and quantify bacteria with FACS, however in spite, it is supposed to work (Previous studies and In silico analysis of the probes), I only get high fluorescence but unspecific hybridization.I have tried different variables to increase the specificity:-Formamide concentration in the Hyb buffer (20mM Tris 7.0, 0.1% SDS, 0.9M NaCl)-Increase wash steps and change pH (SSC20x)-Change hybridization temperatures (From 48° to 70°) 4 hrs with agitation-Reduce the concentration of the probesProbes:Universal GCT GCC TCC CGT AGG AG- 6FAMBifidobacteria GAT AGG ACG CGA CCC CAT-CY5Lactobacillus ACA TGG AGT TCC ACT-CY3In silico analysis: MathfishAll the bacteria are pure cultures washed in PBS, fixed in Alcohol 50% or formaldehyde 3.7% and the permeability of the bacteria have been tested with PIHowever, none of these changes had a significant change. Is there anything else that I could try?Thank you in advanceRelevant answerFalah Hasan Obayes Al-KhikaniJul 30, 2020Answer Good question View16 Recommendations Rola H Aliasked a question related to Fluorescence In Situ HybridizationAnyone has tried the ThermoBrite Elite FISH Auto Slide Prep?Question8 answersDec 3, 2014The ThermoBrite Elite system automates the pre- and post-hybridization steps in FISH testing.How efficient is it? Is it a closed system? Is it FDA approved?Relevant answerDora Elisabeth DiazFeb 7, 2020AnswerAlguien ha probado el Xmatrx® NANO de Biogenex?View0 Recommendations Deven Patelasked a question related to Fluorescence In Situ HybridizationFISH probes without any fluorophores or Biotin/DIG attached to it?Question2 answersJan 14, 2020Hi. I am looking for a FISH probe that does not have any fluorophore or DIG/Biotin molecule attached to it. Instead I need some inactive protein like Cas-9 (Or any other available protein) attached to it. If that s possible to do then Is it available anywhere? If do then please suggest.Thank you.Relevant answerDeven PatelFeb 6, 2020AnswerHi Sreemukta. Thank you for your interest in my question and for your reply.I did try fluorophore attached as well as DIG/Biotin attached probes but the problem with DIG/Biotin probes is that it has lot of background signal and fluorophore attached probe does not have strong signal. So I was curious if something like I proposed in my question is available so I can use primary antibody to detect the protein attached to the probe and then do conventional assay to detect the signal.View0 Recommendations Sana Nasimasked a question related to Fluorescence In Situ HybridizationIn situ hybridization on cre-recombinased mice protocol?Question3 answersJan 8, 2020Has anyone done FISH on cre-recombinased mice? I want to know if anyone has successfully been able to quench the autofluorescence from the those mice to successfully visualize the targeted genes? I have cre-mice crossed with ROSA26 tomato mice that I would like to do FISH on. Any help or guidance would be greatly appreciated. Thank you!!!!Relevant answerVincent SimonJan 14, 2020AnswerHi Chrisopher,I had the same problem with a couple of probes that worked separately but not together. I solved the problem by increasing the dithiothreitol (DTT) concentration in the hybridization mix up to 1M. Maybe this can help...View0 Recommendations Marina Stolyarasked a question related to Fluorescence In Situ Hybridization What is the problem with the DNA probe?Question3 answersDec 8, 2019Hello. We are trying to make a DNA probe for FISH. But fluorescent signals are observed at the periphery of the nuclei (see figure). What could be the problem? The probe was made using DOP-PCR from a BAC clone. I give an example of the obtained product on agarose gel electrophoresis of 1.5%. After DOP-PCR (with Cy3-dUTP), the product was purified and a hybridization buffer (70% formamide, 10% dextran sulfate, 2X SSC, water) was prepared in which 5 μl of purified amplicons were dissolved. The resulting probe was applied to a glass slide with the nuclei of human leukocytes. Denatured at 75°C for 5 minutes, then hybridization overnight at 37°C. The next day, they were washed first in a 0.4x SSC in a water bath at 73°C for 3 minutes, then in 2X SSC with Tween 20 for 30 seconds, dried and examined under microscope with a DAPI /Antifade.FISH Cy3 2.jpg27.09 KBизображение_viber_2019-12-07_00-40-45.jpg16.66 KBRelevant answerPrit Benny MalgulwarDec 11, 2019AnswerIt looks more like a speck which has acquired the fluorescent. Did you confirmed whether its amplifying the right DNA segments, probably doing a sequencing or restriction digestion of the probe sample. Another thing you could be possibly tune the hybridization condition. If its GC rich region try doing till 80degree and 37 degree ON incubation. Further, did you tried adding any control probe, CEP are the best to go. View0 Recommendations Mohammad WAQQAS Mirzaasked a question related to Fluorescence In Situ HybridizationI want to detect Anammox bacteria in my sludge sample, So please suggest me from where i can do FISH (Fluorescence in situ Hybridization) Analysis?Question2 answersFeb 17, 2019Following are probes required for fish analysis 1. AMX3682. EUB338, EUB338II, EUB338III3. NSO1904. NIT35. Nsv443Relevant answerJosé Henrique Pastorelli JuniorDec 2, 2019AnswerI used in my research the 16 Rna technique. There are many papers that you can see it.View0 Recommendations Piet van Vlietasked a question related to Fluorescence In Situ HybridizationWhat causes strong surface signal in WISH ?Question6 answersApr 4, 2015HiI ve been doing whole mount ISH on E9.5-10.5 mouse embryos (with DIG-labeled RNA probes). For a long time, I ve been struggling with getting a decent signal before the background starts to become too strong (NBT/BCIP). On gel electrophoresis, the RNA probe seems ok, so I m assuming the probe quality is ok and I ve included several steps to reduce background (levamisole, acetic anhydride in TEA, RNAse).After sectioning some embryos that were hybridized for a cardiac specific probe and showed a signal in the heart as well as some overall background, I found some weak expression in the heart, but also a very strong signal all over the embryo surface.Has anyone seen this before? I ve googled google extensively, but I have not been able to find a convincing answer. I suspect it may be residual background signal, but am also considering overfixation/suboptimal protK treatment causing suboptimal penetration of the probe (even though I do a 9-10 minute incubation with 10 ug/ml prot K at room temperature). Thanks.Relevant answerLeiming YouNov 8, 2019AnswerMaybe, you can try the longer and more specific RNA probes for your target mRNAs.View0 Recommendations Michelle Klesseasked a question related to Fluorescence In Situ HybridizationWhat is the optimal pretreatment for fluorescence in situ hybridization on human skin samples?Question4 answersOct 11, 2019I am currently trying to do FISH on human skin biopsies that have been routinely fixed in formalin and embedded in paraffin. I am using the CEN X/Yq12 probes from ZytoVision and their ZytoLight Tissue Implementation Kit. De-paraffinization was in xylol, isopropanol, 96% and 70% ethanol. The manufacturer s protocol performs a heat pretreatment for 15 min at 98°C in a citrate buffer and continues with a pepsin treatment at 37°C which should be optimized for the tissue and probes. Denaturation was at 75°C in the dark. Hybridization was at 37°C overnight. All (wash) buffers were used from the kit at correct indicated temperatures.I saw a positive signal for the X probe with 6 minutes of pepsin in a female tonsil test sample. 6 minutes seemed to have also worked for the Y probe in one male skin sample, but no signal for either the X probe or a second skin sample from the same run. Since then, I have tried 2, 4, 6 and 8 minutes of pepsin in more skin samples but haven t been able to get any signal. A second tonsil sample (male) also failed to give a signal with 6 min.Nuclear counterstain was with ProLong Diamond or VectaShield. Additional CD3 staining worked fine in all cases. X and Y probes are both in the same tube (dual color probe), so it s not possible that I forgot to pipet one. Errors in microscope/laser/filter settings are rather unlikely. Thickness is 2 µm as recommended.I am desperate for any advice on what could be issue here. I can still try to reduce the heat pretreatment or play around with the pepsin incubation, but I m running out of time. Any theory or idea for troubleshooting will be highly appreciated!Relevant answerConnor ScottOct 16, 2019AnswerThe most likely issue is that your nucleic acid is crosslinked to much - this is normally due to excessive length in formalin. You may want a more aggressive antigen retrieval, you could heat for longer if needed - we heat for 10 mins @ 121C - as long as morphology doesn t suffer you could be okay. You could also try using a high pH buffer like Tris instead of sodium citrate. Another approach would be to use a microwave or formic acid to uncross link your samples. Fixation time may vary for sample to sample, but your target probe will also vary by how much it is affected by fixation, its annoying but you need to try and find a good balance.View24 Recommendations Shruti Guptaasked a question related to Fluorescence In Situ HybridizationWhat are the maximum number of hours to keep intestinal tissue/skin samples in 4% formalin for fixing?Question7 answersAug 26, 2019Hello fellow scientists I am planning to do fluorescent in situ hybridization work on fish intestinal tissue and skin samples. For which, I will be fixing my samples in 4% PFA for 12 to 24 hours. The problem is; someone is doing the sampling on behalf of me and therefore I won t be able to process my samples right after fixing. They will be shipped to me which will take 2-3 days.My question is, is it okay to keep the samples in formalin for 2- 3 days. Will this effect my hybridization work?Any tips will be appreciated!Thank you!Relevant answerOlaf J.F. SchreursAug 29, 2019AnswerSomething that is not mentioned yet is that you actually can underfixed tissue as well. I have tested small biopsies that were fixed 16h or less, which did not stain optimal in ISH, even they worked fine in IHC. It gave higher variability and more unspecific staining. I discussed it years ago with the old Ventana technical staff, and they had similar experience. For ISH, 1-2 days is optimal, 2d leads to diminished signals, 1d leads to higher background and uneven staining.View21 Recommendations Haixin Jiangasked a question related to Fluorescence In Situ HybridizationHow to make Eubacterial cell wall permeable when prepare for FISH?Question4 answersMay 31, 2019In the protocol of the book, FISH Handbook for Biological Wastewater Treatment, writed by Jeppe Lund Nielsen, the sample is usually fixed independently for both Gram negative and Gram positive cells. However, Eubacteria includes Gram negative and Gram positive bacteria, and I want to identify both of them in one microscopy observation. Now, should I fixed ram negative and Gram positive bacteria separately?Relevant answerKhaled KhleifatJun 4, 2019AnswerI think the below reference is helpful Malanovic, N., Lohner, K. (2016). Gram-positive bacterial cell envelopes: The impact on the activity of antimicrobial peptides. Biochimica et Biophysica Acta (BBA)-Biomembranes, 1858(5), 936-946. View20 Recommendations Yiyu Mengasked a question related to Fluorescence In Situ HybridizationHow to make the cells less sticky for flow FISH?Question1 answerFeb 22, 2018I am currently optimising a flow FISH protocol for an archaea species. The cells are fixed with and final concentration of 50% (v/v) ethanol before FISH hybridisation. The FISH protocol is carried out in cell suspension/pellet, and the resulted pellet is suspended using 0.5 mm needle in buffer solution prior to microscope check. There are several problems: 1) the cells tends to form aggregate;2) the majority of archaeal cells are not labelled with the probes; 3) there is a very strong fluorescence background when checked under the microscope;My assumption is that, the extracellular material of the archaeal cells is very stick, and this makes the cells get burst when physical force is applied. There might be other explanations. Can anyone offer some advice? Thanks in advice.YiyuRelevant answerMirna SekulićMay 28, 2019AnswerHello! Were you able to find a solution? I m experiencing similar problems.View0 Recommendations Cheng Xuasked a question related to Fluorescence In Situ HybridizationHow can I do DNA FISH on a short region (~1kb) in the genome?Question3 answersApr 26, 2019Hi! I m new in the field of Fluorescent in Situ Hybridization (FISH).I want to use DNA FISH to visualize a small region in the human genome (around 1kb). I m not sure if this is too short to use probes generated by nick translation. I guess I probably need to order a set of short probes that all anneal to this region to enhance my signal. Does the Stellaris® RNA FISH system from Biosearch Tech (https://www.biosearchtech.com/products/rna-fish) apply to my case?Or do you think this experiment is doable? What is the best way to do it?Thanks a lot!Relevant answerAnastasia NaumenkoApr 28, 2019AnswerHi Cheng,As I recall BACPAC offers construct design.Well, maybe PNA FISH would be the best option for you then? In general, peptide nucleic acid probes enhance the signal greatly. Check out this paper Article Fluorescent in situ hybridization (FISH) – The next generation and this resource for custom PNA synthesis http://www.panagene.com/_ENG/html/dh_product/prod_list?cate_no=3 View0 Recommendations Omer Goldbergerasked a question related to Fluorescence In Situ HybridizationRNA FISH and protein immunofluorescence in bacteriaQuestion1 answerApr 4, 2019 I am trying to combine the two methods of RNA FISH and protein immunofluorescence to one protocol and apply it on E. coli cells.Does anyone have experience in such procedure in bacteria?Anyone has a protocol or recommendation?Relevant answerDaniel StantonApr 16, 2019AnswerWe use a more specific kit called RNAscope. It gives a higher signal amplification. They promote teh use of secondary staining such as H E and we have used Toludine Blue O. I have not combined immunofluorescence, but as long as there is not an ethanol step used on slides developed with a kilometric kit, you should be fine. The alcohol decolorizes the slide. They do make fluorescence RNAscope kits as well. It is worth a try but you will likely at least have great FISH results.View0 Recommendations Amraoui Hajarasked a question related to Fluorescence In Situ HybridizationDoes the position (5 or 3 ) of the fluorochrome Cy5 is important for a Fluorescence in situ hybridization method ?Question2 answersApr 3, 2019Does it impact the hybridization on the DNA target ?Does it impact its stability ?Relevant answerJuan I. Montoya-BurgosApr 15, 2019AnswerDear Amraoui, The fluorochrome Cy5, when positioned in 5 end, can slightly improve the stability of the duplex as compared to the 3 end. Here is a reference explaining this property: Article Cy3 and Cy5 dyes attached to oligonucleotide terminus stabil... Best regards.View3 Recommendations Patrick Weberasked a question related to Fluorescence In Situ HybridizationCan someone provide some protocol for the combination of ImmunoFISH + Click-it chemistry?Question4 answersJun 24, 2013I want to combine FISH protocol with EdU Click-it chemistry as well as immunofluorescence. Does anybody have a protocol or idea in which order to combine these different staining methods? Are there any specific treatments necessary? At least for immunofluorescence + FISH I know how to handle it, so the question mainly concerns the combination with Click-it reactions.Relevant answerTaras PasternakFeb 19, 2019AnswerYou also can try F-ara-EdU to avoid toxic effect. FISH definitely must be used before click-IT chemistry!View0 Recommendations Jorge Enrique Vazquez Buchelliasked a question related to Fluorescence In Situ HybridizationDo FISH (Fluorescent in situ hybridization) probes need a quencher? Question3 answersDec 31, 2018Can I only label mltiple intern sites in the probe?Relevant answerKaren A. DarbinyanDec 31, 2018AnswerHello, normally FISH technology not require quenching but oligonucleotide probes labeled with fluorescent dyes are used in a variety of in situ applications to detect specific DNA or RNA molecules and in some cases probe fluorescence might be quenched upon hybridization in a sequence specific way. View6 Recommendations Taylor Stimpsonasked a question related to Fluorescence In Situ HybridizationHow to permeabilize free floating mouse brain tissue for dual probe in situ hybridization?Question6 answersOct 31, 2018Hello, so I am doing an experiment where I am trying to locate specific transcription factors in the mouse brain. I have decided to use a dual probe fluorescent in situ hybridization kit that I ordered from a company. However, the protocol that the company provided starts at the hybridization step, but I need to know how to permeabilize the tissue. I am doing the procedure as free floating tissue at 30um thickness and was wondering if anyone had any ideas on how to permeabilize the tissue? So far I have not received any specific fluorescence and I believe it is because I am not permeabilizing the tissue enough.Relevant answerSnow NieNov 22, 2018AnswerYou can try to add some Triton X 100 before you do FISH,View0 Recommendations Mikael Nikuasked a question related to Fluorescence In Situ HybridizationWhat is the most powerful fluorescent detection system for IF and FISH?Question6 answersNov 7, 2018I m looking for solutions to maximise the signal-to-noise ratio in immunofluorescence and/or FISH. We are routinely using Alexa Fluor labelled secondary antibodies but now we need something more powerful. We primarily work with formalin fixed tissues, so the color should preferably NOT be green (due to autofluorescence).Relevant answerVivica GrotelueschenNov 16, 2018AnswerAs a target is typically bound by several antibodies, it might help to enlarge your detction sandwich . For example, if you have a human-anti-XYZ antibody as primary ab, use an unlabelled rabbit-anti-human in the second step and a labelled goat-anti-rabbit tertiary antibody for detection. It might help, but might also result in more background signal as all existing signals get multiplied. View12 Recommendations Sebastian Eulerasked a question related to Fluorescence In Situ HybridizationDetermining microbe/prokaryote diversity in field experiments [Microbiology / microbial ecology]? Question13 answersOct 15, 2018 Hi fellow researchers,I have recently begun a PhD in an interdisciplinary team that involves some microbial ecology in field experiments on my part.Correspondingly I am looking for a way to determine especially prokaryote diversity from soil and water samples (types of species and relative abundance basically) in a mobile setup.It should be relatively quick and especially not too expensive to buy up-front.I am not sure about FISH as the technique seems to be more about looking at the occurence of specific strains that you already know before-hand (and thus know the complementary DNA).PCR techniques use quite a lot of rather costly equipment and is pretty lab-bound to my experience.The abundance itself could be achieved by DAPI/Hoechst staining and fluorescent microscopy.Any way to get a sense of types of prokaryotes involved / general diversity in the field though?May alternatively look at fungi in the field aswell so if that would work in a similar fashion that would also be great.Any ideas would be much appreciated.ThanksRelevant answerDeni RibicicOct 16, 2018AnswerHi Sebastian,I would have immediately suggested analysis of 16S rRNA from environmental DNA by employing NGS, e.g. Illumina MiSeq. However, this is a PCR based technique. Nevertheless, any sequencing laboratory will do downstream operation after DNA has been isolated from the sample. I think you could even send a raw sample to them and they would do DNA isolation and the rest. In the end you would get a qualitative and quantitative report on the microbiome of interest. Of course, this kind of complete workflow and analysis comes with a price, you should ask for a quote to calculate your expenses and to check whether is it in line with your budget. Alternatively, you can perform part of the workflow alone to lower the costs, e.g. isolate DNA, and analyse sequences coming from he sequencing laboratory.Another method that you could use for a rapid quantitative and qualitative analysis of your microbiota are the microarray techniques, which don t require PCR at all. Does a Phylochip ring a bell? If not, check it out (https://ipo.lbl.gov/lbnl2229/)! I am not sure about FISH as the technique seems to be more about looking at the occurrence of specific strains that you already know before-hand (and thus know the complementary DNA). Exactly, if you want to assess total microbial community composition FISH technique is not the way to do it. The abundance itself could be achieved by DAPI/Hoechst staining and fluorescent microscopy. Again correct, but you will lack compositional information.To obtain both, qualitative and quantitative information of total microbial community in certain environment, in my opinion, your best bets are molecular techniques mentioned previously. I would refrain myself from staining approach in this instance.Hope this helps.DeniView10 Recommendations Meng-Ju Wuasked a question related to Fluorescence In Situ HybridizationHow could I stain extrachromosomal DNA by fluorescence in situ hybridization (FISH) ?Question4 answersAug 30, 2018Hi everyone, Is there anyone who has experience on staining extrachromosomal DNA by fluorescence in situ hybridization (FISH) ? I would like to stain extra oncogenes on extrachromosomal DNA and chromosomal DNA. Could you share your protocol? I really appreciate your help!Relevant answerSonal Rajiv BakshiSep 6, 2018AnswerDear Meng-Ju Wu, Are you dealing with human constitutional samples, is the information on origin of marker chromosome available? in that case you can select BACs corresponding to that region, make FISH probes, hybridize and observe, methods for this are on [https://www.e-c-a.eu/en/WORKING-GROUPS.html]. If you do not know the origin of the extra DNA material, and want to stain with FISH, see the link for marker chromosome on above link, and contact @Dr. T. Liehr for methodology details. Hope this is useful.View5 Recommendations Peter Poczaiasked a question related to Fluorescence In Situ HybridizationDoes anybody know a lab in Australia or New Zealand who carries out in situ hybridization, FISH, GISH with polyploid plants? Question1 answerFeb 23, 2018Does anybody know a lab in Australia or New Zealand who carries out in situ hybridization, FISH, GISH with polyploid plants? Relevant answerPudji WidodoAug 23, 2018Answer I think in Wellington Regional Genetics Laboratory, CCDHB New Zealand View0 Recommendations Azamo Gaston-Giberinasked a question related to Fluorescence In Situ HybridizationHave someone already performef Fluorescent in situ Hybridisation with PBMCs ?Question4 answersJun 18, 2018 Hello everyone,I isolated PBMCs for Fluorescent In Situ Hybridization but I also have more Background when performing FISH. can some one tell me what currently contamination sources can occured when doing Ficoll Extraction form PBMCs?.In my case I suspect a contamination with thrombocytes or Erythrocytes it that true? when true how can I purified my sample of PBMCs? Relevant answerAzamo Gaston-GiberinJul 13, 2018AnswerThanks all of you to yours contribution. I would try to put in application all these suggestions.View0 Recommendations Robert Cody Sharpasked a question related to Fluorescence In Situ HybridizationProblems with fluorescent in situ hybridization (FISH) for MycobacteriaQuestion6 answersAug 10, 2017Hello all,I am having problems with FISH using DNA probes for staining mycobacteria (specifically Mycobacterium avium subspecies paratuberculosis). It appears that about 25% or less of the time the probes are actually going into the bacteria and don t look that bad (but I have to put the smart gain on max just to see color). The other half of the time the probes seem like they are not penetrating through the cell wall. I have tried many times changing our protocol, but still only getting that percentage of actual success. Even when using the same protocol over and over, the results still come out different almost each time. Other bacteria species work pretty well, it s just Mycobacteria that are not working well. I know Mycobacteria have the thickest cell wall of almost all bacterial species, but the protocol that I use still should work.  Has anyone worked with bacteria (specifically mycobacteria) using FISH using DNA probes (alexa probes are added to the 5 end)? Please let me know your protocol and I will show you ours as well. Thanks. Relevant answerAna MoriJun 18, 2018AnswerHello Cody Sharp, I m working with RNA probes for Mycobacterium since last year.I use clinical samples of swine lymph nodes, we use tissue paraffin-embedded, that are PCR positive for Mycobacterium avium, or M. bovis, or M. tuberculosis. I already used Lyzozyme, Achromopeptidase and Tripsina 0,1%. Nothing worked. The Miguel s protocol worked? I m sending to you a picture of our FISH, and this is the only fluorescence that we got. We think this are the macrophage granules. not Mycobacterium. The fluorescen is in the health tissue, not in the necrosis. Do you have some news, and better results? Snap-42.jpg575.21 KBView0 Recommendations Małgorzata Blatkiewiczasked a question related to Fluorescence In Situ HybridizationFISH on frozen section?Question6 answersJun 8, 2018Hello, I m going to start my journey with Fluorescent in situ hybridization. Do you have some good protocol to FISH for miRNA on frozen human liver section?Thanks for all!Relevant answerWolfgang H. MussJun 14, 2018AnswerDear Małgorzata Blatkiewicz From the ResearchGate archives/Publications:[just always search full text via SEARCH Upper menue line in ResearchGste (insert keyword, search phrase ore sentence(s) - click the loupe-icon and - after some second of waiting - choose from the sections: Authors or Projects or Publications or Questions ]. Naturally you can search also in Scholar Google (publications as well as recipes) or GOOGLE and any other data base (like PMC/NCBI/PubMed)Here you go - to start with: Article LNA-FISH for detection of microRNAs in frozen sections Article Documentation of EWS Gene Rearrangements by Fluorescence In-... Article A modified immunofluorescence in situ hybridization method t... https://www.molecular.abbott/int/en/vysis-fish-knowledge-center/frozen-tissue-prep-for-fish : Standard ProtocolCut 5um sections on silanized or positively charged slidesDry at room temperature for 15-30 minFix in 4% formaldehyde in PBS at 0 ºC for 15 minWash with PBS and start the Paraffin Pretreatment kit protocol at the .2N HCl step and follow through to the endAlternate ProtocolCut sections as before and dryFix in Carnoy s fixative (3:1 methanol: acetic acid) for 5 min.Begin the Paraffin Pretreatment kit protocol just prior to the Protease step (Wash Buffer for 5 min.)and last but not least: Preparation of Paraffin Sections and Frozen Tissue for FISH 2006 https://ccr.cancer.gov/sites/default/files/preparation_of_paraffin_sections_and_frozen_tissue_for_fish.pdf(no warranty for simplicity, easiness or guaranteed success, but - as always - confident in orthers former tremendous work and scientific spirit).View10 Recommendations Darellynn Ooasked a question related to Fluorescence In Situ HybridizationFISH Analysis Guidelines for CLL?Question4 answersJun 8, 2018Dear Researchers,I need a comprehensive guideline to follow to determine analysis cut-off points (e.g.: number of nuclei per sample, percentages to consider a deletion present, false +/- testing for each probe since the % of hybridisation is different for each probe).I am using Leica: TP53, LEU1, +12 and ATM mutations for my FISH experiments. So far the only one I have found that is of any use is this:Article Guidance for Fluorescence in Situ Hybridization Testing in H... Does anyone have any other papers that may be useful/helpful for my purpose? Would be better if it was specific to CLL. Thanks in advance.Relevant answerSharafaldin Al-MusawiJun 11, 2018Answer Hi dear Darellynn Oo I think the following link can help you;Best and kind regard.https://www.onclive.com/publications/oncology-live/2011/december-2011/fish-testing-guides-treatment-of-cll-new-drugs-on-horizonView7 Recommendations Piet van Vlietasked a question related to Fluorescence In Situ HybridizationWhat causes ISH/X-gal signal loss during embedding?Question12 answersApr 4, 2015HiI ve been doing WISH and X-gal stainings on E9.5-12.5 mouse embryos.After obtaining a decent (ISH) or even strong (X-gal) signal in the whole mounts, I fix the embryos o/n (or up to several days) in 4% pfa, dehydrate them to 70% ethanol in PBS for a couple of days to improve signal/background ratio, take pictures, and embed them (ethanol dehydration, xylene, paraffin). During sectioning, the signal still looks ok. Unexpectedly, the signal nearly dissolves in the water bath when I stretch the sections. By the time I place the sections on the slides, the signal has become very weak and sometimes nearly undetectable. I ve read some suggestions that incubation in ethanol or xylene may dissolve the color crystals, causing them to leak from the sections, but I have been unable to find exact times. Does anyone know how long is too long? Would any other dehydration method work better with regards to preserving signal?Thanks.Relevant answerPiet van VlietJun 11, 2018AnswerHiBelow is the entire protocolmy colleagues and I use now for X-gal and that seems to work. As far as I can tell, it was the xylene steps during the embedding. I have now shortened those to 5 minutes each, as opposed to the traditional 15-60 minutes (depending on tissue size, I mostly do mouse embryos). Still seems to work, albeit that you will have to check for possible artefacts, because if the tissue is not incubated long enough in xylene, they may not take up the paraffin as well and then they won t section as well. For the few in situs I tried after my initial posts it seemed to have worked too, but I cannot be 100% certain since I only did a few of them. But since both procedures produce crystals, I suspect that the xylene affects them more or less similarly. I have attached a link to a lacZ bible I found online. Could not trace back the original author, but I did find that Andy Groves at Baylor College of Medicine had used the same protocol (https://www.bcm.edu/research/labs/andrew-groves/protocols). He was very helpful when I contacted him. Other than that, please see http://wmc.rodentia.com/docs/lacZ_bible.html, PMID: 28669819, and PMID: 1742021. Hope this helps.Best,Piet - fix embryos in 2% pfa, 0.125% glutaraldehyde in PBS on ice (5 forE9-10, 10 for E11-12)- wash cold PBS- I then keep them in PBS with 0.025% sodium azide at 4C if stored formore than a couple of days- prepare and stain the whole mounts as described in the Lac Z bible link: http://wmc.rodentia.com/docs/lacZ_bible.html  I haven t beenable to track down the original author, but did contactAndrew Kelton Groves, who mentions it in one of his publications. Asan alternative to the subsequent paraffin embedding, they dogelatin/sucrose embedding, which avoids the xylene requirement(protocol and additional explanations attached below). Unfortunately,our cryostat does not go below -22 C, and you need -30 to keep theblocks frozen, otherwise they become more like jelly and areimpossible to section).I always make the staining solution fresh and use X-gal (dissolved inDMF) that has not turned too yellow yet.- I combine several embryos in PBS in one 5 ml glass vial (Fisher) ona rotator (individual embryos marked by removing e.g a limb or tail)- for E11 and older, make sure to puncture the thorax and abdomen toensure proper incubation- rinse 2x 30 -1h and then overnight at 4C on a rotator- stain overnight at 37 C with pre-warmed staining solution (make surethe crystals are dissolved well) on a rotator inside an incubator- post-rinse for 10-15 at 37 C- the staining solution can be used multiple times when kept at 4C andfiltered, but it does tend to stain weaker each time.- wash 3x PBS at RT- fix in 4% pfa/PBS overnight at 4C on a rotator (or longer, sinceoverfixing does not negatively affect the paraffin sectioning).- wash PBS and dehydrate to 70% ethanol for long-term storage orfurther embedding. I also take pictures when they ve been in 70%ethanol since it improves the contrast between embryo and staining.For embedding:- wash PBS, dehydrate 30-50-70 on day 1 (if not already done so above)and then on day 2 80-90-95-100-100% ethanol/water (I normally do 20-30minutes depending on stage)- max 5-10 minutes each (for up to E12, I haven t tried older stages):incubate 50/50 ethanol/xylene, 2x 100% xylene at RT and then 50/50xylene/paraffin and 3x paraffin at 60-62C.For sectioning:- I do 10 um sections, stretch well in waterbath, place on slides, drywell (overnight or longer).For staining:- rehydrate: 2x xylene (up to 5 minutes seems to work very well) then1 minute each ethanol and other step. I ve found that most, if notall, of the remaining staining will keep during these steps. Gentlydipping every 10-15 seconds helps too, but be careful because sectionsmay partially detach.- counterstain with nuclear fast red (my preference) or eosin, anddehydrate again. Mount with Permount. Dry for several days.If you just want to look at whole mounts, they do not need to beoverstained, but just up to the point where it s strong enough andthen fixed. I can get you a clearing protocol from a colleague who isdoing X-gal followed by clearing to look inside the whole mount. Youwill lose some staining however, so it s a bit of trial and error ( PMID: 27348591).View0 Recommendations Marlonni Araujoasked a question related to Fluorescence In Situ HybridizationFluorescent in situ hybridization works for dsRNA virus but not for ssRNA virus. why?Question2 answersMay 22, 2018I have been working with fluorescent in situ hybridization on paraffin-embedded tissue of papaya leaves in order to localize two RNA viruses.After several adjustments in the protocol, the hybridization for dsRNA virus works well, but not for ssRNA virus.My probes have 400bp and was synthesized by random primer labeling with fluorescent dUTP. The template sequence for the reaction was obtained by cloning PCR fragments from the viruses in pGEM-T easy vector. Hybridization temperature, proteinase K concentration, and the denaturation process were adjusted.Does anyone have a possible explanation?Relevant answerKevalkumar ShahMay 25, 2018AnswerHi,Possible reasons for these1. Your ssRNA does not match with your probe2. It may contain low GC and melt at relatively low temperature3. No. of nucleotide that match with probe are not enough for fluorescenceView0 Recommendations Mikael Nikuasked a question related to Fluorescence In Situ HybridizationWhat is the most effective detection system for 16S FISH?Question1 answerApr 10, 2018We are setting up 16S FISH for animal tissues with low bacterial abundances. What is the most effective specific system for probe detection signal amplification?Relevant answerIvan KanchevApr 11, 2018AnswerHave you looked at the ACD Company solutions. They do have quite sick in a good way things in their portfolio.Best,IvanView0 Recommendations Astrid Wendlerasked a question related to Fluorescence In Situ HybridizationDNA in FISH staining looks digested and blurry . What can I improve and what may have gone wrong?Question4 answersFeb 28, 2018The metaphase spreads look absolutely normal outside of the area where the denaturation and hybridisation took place. Within the area of the denaturation and hybridisation, however the metaphases look weirdly shaped and clumping but also the nuclei look somehow digested/degraded.I attached two pictures of the dapi stain to visualise this difference. The probes I am using still work well on the bad looking cells (not shown), but I need clear images with recognisable chromosomes. We already tried reducing the denaturation step and decreased the temperature, but it didn t help.Did anyone come across this issue and can help?Thanks for helping.Best wishes,AstridMeta3dapi.tif5.53 MBMetanice.tif5.53 MBRelevant answerAstrid WendlerMar 9, 2018AnswerThe slides were stored at RT in the lab. It is generally not dry in the lab- I would say it varies. If it would be too humid storing them, would that not also have an influence on the rest of the slide outside the hybridisation zone? The hybridisation takes place in a humidity chamber and there was no sign of the slide drying out.View0 Recommendations Swarnaprava Beheraasked a question related to Fluorescence In Situ HybridizationProblem with FISH(fluorescence In Situ Hybridization) ?Question6 answersDec 14, 2017Hi,I am using EUB338 probe ( 5 - GCTGCCTCCCGTAGGAGT -3 ) at 35% formamide concentration and 46 C hybridization temperature as described by Silva protocol(attached) for bacterial cells on membrane filters. I am also fixing the cells with 4% Paraformaldehyde in PBS prior to hybridization.I am not getting any results while seeing under the microscope. Please suggest in which step I could go wrong or how to troubleshoot.SILVA_FISH_protocols_mono_101025_V2_2.pdf166.76 KBRelevant answerSwarnaprava BeheraMar 6, 2018Answersame cy3 probe? which filter you are using for visualization? View0 Recommendations Peter Poczaiasked a question related to Fluorescence In Situ HybridizationHow to design FISH probes from Solanaceae genome sequence data?Question3 answersFeb 24, 2018I would like to design FISH probes for cytogenetic experiments for Solanum species. There are a number of Solanum genomes available so probes could be ideally done for each chromosome with known locations. I haven t done such thing before, so are there any probes already available for tomato or potato? What should I keep in mind to design probes for in situ hybridization?Relevant answerSeied Mehdi MiriFeb 25, 2018AnswerDear Dr. PoczaiPlease read the following article and I hope you find the answer in it.Comparative Oligo-FISH Mapping An Efficient and Powerful Methodology To Reveal Karyotypic and Chromosomal Evolution.pdf1.32 MBView12 Recommendations Marcos Moresasked a question related to Fluorescence In Situ HybridizationFluorescenc hybridization in situ (Fish) to Mycobacterium spp. Someone has some experience with that? Question2 answersFeb 20, 2018We are trying to break the mycobacterium spp s wall to do FISH in formalin fixed tissues. We already use Lysozime and/or Trypsin, but didn t work. Someone has some experience with that? Relevant answerMarcos MoresFeb 23, 2018AnswerHello Elisa,Thank you for your comments. We are considering to test that suggestions.Best regardsMarcosView0 Recommendations Carlos Alexander Ruiz Pérezasked a question related to Fluorescence In Situ HybridizationHow to reduce bacterial autofluorescence in water samples for FISH/DAPI staining?Question2 answersFeb 14, 2018I have been trying to perform FISH/DAPI staining of bacteria in water samples for cell counts and identification of certain bacterial populations. The issue I am having is that several cells show high autofluorescence in all channels I am using for the probes so I cannot be confident of the signal I obtain with my probes.I have tried using pre-treatments with UV exposure (I read this can potentially damage the ribosome and affect the hybridization) and using Sudan Black B (although this was used in tissues, not planktonic cells) with little success. I was wondering if what protocol/method you know or have used to reduce or at least minimize this issue in water samples? Thanks!Relevant answerDaniel PrietoFeb 14, 2018AnswerDear Carlos, I have no experience in tge kind of samples you are assaying. There are some points however that you may find useful. Provided you are using water samples, it is possible you are handling a lot of photosynthetic bacteria in them. Photosynthetic pigments will of course be absorbing light in several portions of the spectrum and emmiting at some others. Getting rid of them through a selective culture step could help you. In the old times, people would get rid of some autofluorescence issues by incubating 5min with 1% evans blue. This would switch much of the greenish autofluo into the red channel, thus allowing them using Fitc-conjugated stuff. There is still another option which is performing a lambda scanning of your unstained preparation and finding out which portions of the spectra do not autofluoresce. Once identified, you can choose some quantum dots as fluorophores with very narrow absorption and emmission spectra. Hope these comments would help you.Best,DanielView0 Recommendations Evangelia Tachmatzidiasked a question related to Fluorescence In Situ HybridizationWhat rubber cement could I use in FISH experiments?Question11 answersNov 8, 2016Dear all,I am trying to conduct fluorescence in situ hybridization (FISH) experiments and I need a rubber cement for the hybridization step. Do you have any suggestions of what rubber I could use?Relevant answerSaurabh PriyadarshiFeb 9, 2018AnswerElmer s No-Wrinkle Rubber Cement works fine for me and some other labs which i know.View3 Recommendations Esther Mezhibovskyasked a question related to Fluorescence In Situ HybridizationCan I perform FISH staining on previously stained intestinal segments with IF (mounted with gold prolong antifade)?Question2 answersDec 7, 2017The goal is to preserve the mucous layer. The slides have been IF stained for a week now. Relevant answerEsther MezhibovskyJan 14, 2018AnswerThank you Elliot! View0 Recommendations Mona N. Husseinasked a question related to Fluorescence In Situ HybridizationWhat is the method for design oligonucleotide probe for fluorescent insitu hybridization? did i should make gene cloning to confirm probe sequence?Question2 answersJan 11, 2018if i designed a pair of primers and examined their specificity to the exact gene and by PCR i obtained the exact band. can i use this antisense primer as oligonucleotide probe directly for performing fluorescent in situ hybridization or i should make gene cloning and sequencing before using it.Relevant answerMona N. HusseinJan 12, 2018Answerthanks a lotView0 Recommendations Satyajeet Salunkheasked a question related to Fluorescence In Situ HybridizationCan any one provide protocol for design of DNA fluorescent probe for mRNA detection?Question2 answersNov 8, 2017I want to see localization of mRNA in cell using DNA probe there r two alternative LNA probe and DNA probe,I want to know how we can design a DNA based fluroscent probe for mRNA detection ,if any one has used it kindly share protocolRelevant answerWilliam Richard WebbNov 22, 2017Answerif using human cells try smart flare by milliporeView7 Recommendations Tamar Tsertsvadzeasked a question related to Fluorescence In Situ Hybridization I need any advice on Fluorescent in Situ hybridization?Question6 answersNov 6, 2017I am struggling with The XL DLEU/TP53 locus-specific probe (metasystem) detects deletions in the long arm of chromosome 13 and in the short arm of chromosome 17 in Chronic lymphocytic leukemia (CLL) by FISH Relevant answerTamar TsertsvadzeNov 17, 2017AnswerDearWojciech WitarskiI done Fish according my protocol ( metasystem)but at last when i look slide under the microscope have not get result, I think Dapi didn t paint cellscan you help me?What can be reason?What could be the reason that the dipi can not contain cells?View0 Recommendations Alejandro Laudicinaasked a question related to Fluorescence In Situ HybridizationAny Fluorescent Cytoplasm Staining dye?Question5 answersNov 7, 2017I need to stain cytoplasm with a fluorescent dye in order to see it along with a FISH reaction.Relevant answerPatrick KilloranNov 8, 2017Answerthis might be helpful https://www.thermofisher.com/za/en/home/life-science/cell-analysis/cell-structure.htmlView10 Recommendations Tzach Aumanasked a question related to Fluorescence In Situ HybridizationDoes anybody have experience with ImmPACT Vector Red Alkaline Phosphatase (AP) Substrate ?Question3 answersMay 10, 2016Im trying some new reagents for FISH. Fast red dsnt seem to work at all, ant this product from Vector labs claims to be much better- But it looks like it works even worst then the fast red. I cant even get background signal.Any advice?Thanks!Relevant answerTzach AumanOct 25, 2017AnswerHi, thanks for the comments.--- Well, It turns out the substrate was bed. We got a new batch wich worked wonderfully. Actually, It works much better than the ROCHE fast red tablets we used to work with.View0 Recommendations Mc Susannaasked a question related to Fluorescence In Situ HybridizationIn situ hybridization: opening the nucleus?Question4 answersOct 21, 2017During in situ hybridization of DNA usually detergent are used to make pores in the phospolipids containing nuclear membrane. However, in case you perform ISH on FFPE tissue sections containing cells with a nucleus of at least 10 micron (e.g. cancer cells), which are cutted around 3-5 micron, I would suppose that most cells are already open? Why is then supposed to be so necessary to pretreat your sample very well?Relevant answerGudrun LangOct 21, 2017AnswerPretreatment of FFPE sections is necessary because of formaldehyde fixation and its effect on nucleoproteins (histones) and DNA.Nuclei are cut and open but DNA is in close combination with nucleoproteins, that hide the bindingsites of the probe and they have to be digested by the proteinase. So the probe can bind better. Heat pretreatment reverses the effect of formaldehyde on proteins. Methyloladducts are loosened again and proteins gain almost their native structure. This helps also the proteinase to reach its binding-sites. Digestion after heat pretreatment works more effective.View7 Recommendations Anna Banach-Wiśniewskaasked a question related to Fluorescence In Situ HybridizationIs it any way to deal with autofluorescence of activated sludge?Question3 answersSep 26, 2017At the moment I am working with Fluorescence in Situ Hybridization to study microbial communities in activated sludge Samples which I ve recently studied posses a strong tendence to autofluorescence. I ve tried to deal with this problem by making some procedural modifications or reducing the amount of some extracellular substances. But I am wondering if there are more ways (maybe more effective) to minimalize the autofluorenscence of activated sludge. Relevant answerJason A KilgoreSep 28, 2017AnswerThat s a tough one. I m not a sludge expert, but unlike with cells in culture or tissue samples, you can t easily do a sodium borohydride treatment. So I think your options would be: 1) choose dyes that are outside the autofluorescent spectrum of the sample, 2) image with one wavelength left open (specific label in green, and an empty channel in red) then subtract one image from the other using imaging software to artificially subtract out the autofluorescent signal, or 3) photobleach the sample with very strong UV light prior to labeling in the hope that the autofluorescent compounds will fade and not come back.View5 Recommendations Muhammad Zafar Iqbalasked a question related to Fluorescence In Situ HybridizationWhat would the method to mix biotin- nick translation mix and Digoxygenin-nick translation mix together to get yellow signals?Question1 answerSep 9, 2017I want to mix digoxygenin and Biotin together in order to get yellow signals In FISH ( Flourscent in Situ Hybridization ） assay. Can anyone guide me either I have to mix both together in nick-translation reaction or do nick translation reaction separately and then mix nick translation products together?Relevant answerUte NeubacherSep 15, 2017AnswerHi Muhammad, I mix the nick translations together in the hybridisation buffer as a cocktail and put this cocktail after denaturation on the tissue sections for hybridisation. The detection of the two probes by immunohistochmistry (Anti-Dig and Strptavidin) I do seperatly. Depending on your protocol take care about the different blocking steps you need.View0 Recommendations Nitin Sabherwalasked a question related to Fluorescence In Situ HybridizationHow to attach FACS-sorted patient-derived-xenograft (PDX) cells on glass cover-slips?Question5 answersSep 7, 2017I am struggling to attach FACS-sorted PDX cells onto the glass coverslips, for further downstream experiments like immuno-stainings and RNA FISH. Can someone help?Relevant answerNitin SabherwalSep 11, 2017AnswerThanks Sabine Strehl and Pieter Louwe. These things have not worked for me. I must say I still need to give it a go with gelatin and collagen which I will do soon.View0 Recommendations Muhammad Arslanasked a question related to Fluorescence In Situ HybridizationSelection of embedding media for plant samples to avoid detachment on hybridization slide during washing in fluorescent in situ hybridization (FISH)?Question20 answersAug 11, 2016Hi all, I need expert opinions on this matter as my plant samples are detached during washing step of FISH. I used agarose as well as transparent nail polish for this purpose and to me agarose didn t work at all but nail polish works slightly. It provides a base to sliced plant tissue, when I put my hybridization slide into the washing buffer (50 ml falcon) and then in water bath; after heating up to 48 degree Celsius, the nail polish is no more stick and my plant samples swim in freely in the media. Though I doubt that it may not affect the hybridization but still I am not sure. Can you please share your practical experiences?Thanks in advanceRelevant answerLaith Al-AniAug 17, 2017Answer20https://www.genomicvision.com/Molecular/CombingView0 Recommendations Mehdi Montazerasked a question related to Fluorescence In Situ HybridizationAre there any ways to test an expired FISH probe in order to find if it still works?Question5 answersAug 6, 2017I have some FISH probes which have been expired about 5 years ago. How can I find if they are still usable? I am looking for some way except performing a whole FISH study. Relevant answerSabine StrehlAug 7, 2017AnswerIn my opinion there is no way around testing your probes by conducting FISH experiments.View4 Recommendations Steven Welcasked a question related to Fluorescence In Situ HybridizationExperience using Fluorescent In Situ Hybridization (FISH) on Cells in Suspension for Flow Cytometry?Question5 answersMar 17, 2016Hi, I am interested in doing FISH on leukocytes in a single cell suspension so that they can be analyzed via FACS.  If effective, this would allow for more efficient analysis and of many more cells than traditional FISH-fluorescent microscopy (a method I have previously used with success).  Protocol: 1) Cells fixed with Carnoy s in a drop-wise manner. Spin, decant supe.2) Permeabilize cells with 0.1% Igepal in 2X SSC. Spin, decant supe.3) Resuspend cells in hybridization buffer (70% formamide 2x SSC).     a) Co-denaturation: Expose cells and probe to 75C for 5 together. 37C o/n     b) Separate denaturation: Cells 72C for 5 and Probe 90C for 10 .  37C o/n     c) Separate denaturation: Cells 37C for 5 and Probe 90C for 10 .  37C o/n4) Wash with 0.1% Igepal in 2X SSC. Spin, decant supe.5) Preheat 0.3% Igepal in 0.4X SSC to 72C.  Add buffer to cells expose to 72C for 2 . Add equal volume ice cold PBS. Spin, decant supe.6) Resuspend in PBS.At this point I m not attempting FACS,  I am cytospinning on to slides until I optimize the hybridization conditions.  I have given it a couple passes and I am routinely running into the same issues: 1) Enlarged nuclei, (the nuclei appear to be large and have a more diffuse DAPI signal). The nuclei seem to denature/enlarge once exposed to 70% formamide 2x SSC independent of heat.   Possible problems: inadequate fixation, absence of graded alcohol dehydration steps like in traditional fix.2) Ineffective hybridization.  Since the conditions used have successfully worked for traditional FISH it would seem they should work.  Also probably a inadequate fixation or denaturation issue.3) Cell clumping.  Cellular denaturation is resulting in the netting of cells by DNA strands after exposure to 70% formamide 2x SSC.  I have some ideas of what to try next, but if anyone has had previous experience with Flow-FISH and could provide insight that would be great.Thanks!Relevant answerFabio Henrique Brasil da CostaJul 28, 2017AnswerHey Steven,I know it s been over a year, but if you re still interested, take a look at the PrimeFlow kit from thermo. https://www.thermofisher.com/us/en/home/life-science/cell-analysis/flow-cytometry/primeflow-rna-assay.htmlView0 Recommendations Lola Toomeyasked a question related to Fluorescence In Situ HybridizationUse of 16s rDNA in eucaryote phylogeneticsQuestion3 answersJul 19, 2017Hello,I am getting confused with the 16S marker and would need help.I saw that the 16S rDNA is mostly used in bacteria research and is the component of the 30S small subunit of a prokaryotic ribosome, 18S for eucaryotes. So why is it also used for animal phylogenetics (e.g. fish) ?Also just to be sure, from what I read, 16S rDNA is maternally inherited and non coding, right ? I have many stop codons in my sequences, whatever the reading frame used..If someone can help me I would be very grateful !Relevant answerSalvador Ramirez-FlandesJul 23, 2017AnswerHello Lola,You are right in everything you just said. The 16S rDNA gene encodes the small subunit RNA of the ribosome (ssu_rRNA) in prokaryotes, while the 18S do the same in eukaryotes. However, eukaryotes are composed by more complex cells that includes organelles. One of those is the mitochondria, which has its own genome and synthesize its own proteins, so it needs ribosomes, hence it needs ssu_rRNA. Now, according to the endosymbiotic theory (or symbiogenesis), mitochondria has bacterial origins (in fact the eukaryotic cell was the product of the endosymbiosis of bacteria within archaea -- see attached publications for more details), and that is the reason why mitochondria has a prokaryotic ssu_rRNA (i.e. 16S). This subunit is purely RNA -- it does not encode any protein, which is the reason it can has stop codons within because it is never interpreted in terms of codons.. it is just not translated, it is simply folded as RNA and used as such as part of the ribosome complex.And yes, only the egg cell has mitchondria, not the sperm cell.Relevant scientific literature:Williams, T. A., Foster, P. G., Cox, C. J., Embley, T. M. (2013). An archaeal origin of eukaryotes supports only two primary domains of life. Nature, 504, 231-236.Martin, W., Roettger, M., Kloesges, T., Thiergart, T., Woehle, C., Gould, S., Dagan, T. (2012). Modern endosymbiotic theory: Getting lateral gene transfer into the equation. Endocytobiosis Cell Research, 23.I hope that helps. Best regards.View8 Recommendations Gonçalo Pinheiroasked a question related to Fluorescence In Situ HybridizationIs there a way to remove NBT/BCIP residues from glass slides?Question8 answersMar 29, 2015I am doing ISH in cryosections, and when I develop the staining in drops, or sometimes in a container, some NBT/BCIP residues stick to the slide, and make it harder to get a good picture of the staining, due to the background colouring. Is there a way to wash away the residues, or to prevent them from forming? I have already tried to extend greatly the number and duration of washes I do post-probe labelling, in order to remove all antibody leftover.Thank you for the helpRelevant answerPan LinJul 3, 2017AnswerDid you try 98% ethanol? I found this can wash away NBT/BCIP precipitates very quickly (in five minutes). But I am not sure this treatment will also wash away antibody.View8 Recommendations Yousef Daneshmandpourasked a question related to Fluorescence In Situ HybridizationCan we use QF-pcr for single cell analysis?Question2 answersMay 14, 2017HI.I mean can we use it like FISH that with a software it can calculate percent of aneuploidic cells in samples like sperm?thanks for your comments.Relevant answerYousef DaneshmandpourJul 2, 2017AnswerThank you Houshang Nouri.View0 Recommendations Camila Freitas Stahlasked a question related to Fluorescence In Situ HybridizationHow to preserve cells for FISH?Question5 answersJun 27, 2017Hello! I am going to do FISH and I have to make my experiment soon, but the probes are not here yet. How can I better store the cells and for how long? I saw in one protocol that you can leave in the permeabilizing step with 70% ethanol for 1 week. Is this the best and longest way to keep it?Thank you for the help!Relevant answerluis alberto mendez-rosadoJun 27, 2017AnswerI have do FISH in lymphocytes stored at -20 in fixing solution for a long time without problemsluisView5 Recommendations Susana I Sáasked a question related to Fluorescence In Situ HybridizationFluorescent in situ hybridizationQuestion2 answersMay 19, 2017I need to perform fluorescent in situ hybridization (FISH) in hypothalamic tissue sections.I already have the oligo probes, but they were going to be used in radioactive ISH.Is there a way me to label these oligos for FISH? Are these kinds of oligos only useful for radioactive ISH?Does anyone have some expertise in this subject?Thank you.Relevant answerSusana I SáMay 26, 2017AnswerYes, it helped very much.Gracias...View0 Recommendations Luca Isenmannasked a question related to Fluorescence In Situ HybridizationShort (250bp) mRNA FISHQuestion1 answerMay 17, 2017Hi,I m trying to detect the expression of several 250bp marker RNA sequences using FISH. For this I m free to design the marker sequence but are limited by the size of the construct (200-300bp).My current plan looks like this1) generate random DNA sequences2) Design multiple 20bp probes targeting this DNA using the Stellaris probe designer3) combine those probes into a 250bp marker sequence4) check the probes and sequences for stable hairpin and blast on NCBI balst5) stably integrate my construct containing this marker sequence in a cell line6) detect expression of marker sequence using FISH (several 20bp probes or longer probes)As I don t have any experience in FISH I now have the following questions.-Is it possible to reliably detect the expression of a 250bp sequence using FISH? (just the expression, I m not interested in the location)- I m currently using the Stellaris probe designer and some Microarray probe designer to design my probes. Is there a better alternative or even a curated Database of validated FISH probes?- NCBI blast shows that all the probes designed by Stellaris have atleast 65% homology to random genes. Is this a problem?Many thanks in advance.Relevant answerNoura RamadanMay 21, 2017Answerby FISH, you cant be sure  that the signals you found are from your probe as its length is 250bp, the big problem of Fish is the diffuse, nonspecific signals found after hybridization. you can overcome it by trying techniques for FISH pretreatment that can decrease these problem, also effictive post hyperdization wash method.View0 Recommendations Priyanka kriti Narayanasked a question related to Fluorescence In Situ HybridizationProtocol for Gene loci fluorescence in situ hybridization in suspension cells.Question3 answersMay 10, 2017I am working on gene loci 3-D DNA FISH in suspension cells(primary lymphoid cells), but it s not working and I am not able to troubleshoot.I have tried to execute the experiment many times but I am unable to get the result. Followings are experimental details and queries:Q1- How to isolate BAC DNA. I have isolated BAC DNA by Qiagen large construct kit but genomic DNA  and RNA contamination are still coming.Is any special treatment required for isolating BAC DNA?Q2- I am getting signal along with so much background so just help me out to reduce the background.Q3-I am preparing probe by FISH Tag Kit(Invitrogen) by nick translation but I want to know how much concentration of probe DNA, cot1 DNA you used for precipitation of DNA and in how much volume of hybridization buffer and how many washes is required to get rid of the background? It would be nice if anybody can share the protocol with me. Relevant answerShafqat Ali KhanMay 12, 2017AnswerI am also standardizing Immuno-RNA-DNA FISH and could help you after two weeks I think. But as ann answer of your third question, I would suggest using 10ng/ul probe and 100ng/ul Cot-1 DNA and dissolve it in 20ul of Hybridization buffer (this is for one reaction of FISH). This is not mentioned in the paper but I have got this information straight from the Lee lab. I will upload the detailed protocol tomorrow.Hope this would be helpful.thanksView0 Recommendations Chrysanthi Taxiarchiasked a question related to Fluorescence In Situ HybridizationHow to do in situ (FISH) on cells that are FAC sorted straight onto slides?Question3 answersMar 17, 2017I am FAC sorting testicular cells and I would like to do FISH to tell the spermatogenic stage. Some populations are very rare ~1,000 cells, so I wanted to sort them straight onto slides and do the fixation and staining there (sorting in tubes and cytospin would mean sample loss and the cells would be very dispersed on the slide after that). I have gone through so many protocols that don t really work for the above conditions.. Any suggestion? Relevant answerJennifer RudolphMar 22, 2017AnswerHello Chrysanthi,Maybe you have to try which approach might work better, a fixation with paraformaldehyde before sorting or once your cells are on the slides. You can also do the fixation on the slides, just ensure your cells adhere to it. In principle the protocol for tissues and for cells (on filter) should be the same. Maybe you can have a look here https://www.researchgate.net/publication/246206760_Fluorescence_in_situ_Hybridization_FISH_with_rRNA-targeted_Oligonucleotide_Probes. Still find this work by Pernthaler et al. very helpful.Best regards!Article Fluorescence in situ Hybridization (FISH) with rRNA-targeted...View0 Recommendations Marlonni Araujoasked a question related to Fluorescence In Situ HybridizationHow can I remove the chlorophyll autofluorescence in a fluorescent in situ hybridization protocol?Question3 answersJan 29, 2017I fixed the tissues in paraformaldehyde 4%. The hybridization was made using Alexa Fluor 594 in labeling reaction but I can t confirm if the fluorescence is due to hybridization signal or chlorophyll autofluorescence.Relevant answerMiroslav MacekFeb 2, 2017AnswerI ll not help you, excuse me. Theoretically, chlorophyll could be washed out in ethanol or methanol (that was used several years ago to wash excess of DAPI from membrane filters) but we did not reach perfect results. Moreover, we had even worse experience with picocyanobacteria pigments, which are water-soluble.However, our experience indicates that the fluorescence of such pigments was not the same using different FISH probes. You should take it into account - the error is not the systematic one.The alternative fixatives that could switch off the fluorescence: acid Lugol postfixed with thiosulphate formalin (Jezbera et al. 2005). But not every time (my tool - ciliates- could have more unspecific red fluorescence)Bouin fixative -picric acid in formalin + acetic acid (Fried et al. 2002). But it stains larger object too much to be observed (it is usefull for small ciliates and their food). Generally, the fluorescence is less bright but the background is perfectly black if the preparation is made on membrane filters.Do not hesitate to contact me if you are interested inmmacek@live.co.ukView5 Recommendations Pedro Crevillénasked a question related to Fluorescence In Situ Hybridization¿Which is the typical vacuum pump used for fixating plant tissues?Question4 answersJan 26, 2017I am looking for a vacuum pump to infiltrate solutions into plant tissues in order to fixate them (for molecular biology experiments like ChIP, in situs, etc…).I need something powerful, because it is not just for Arabidipopsis seedlings,  but not too strong and there are plenty of options in the market ¿How much vacuum power do I need?Relevant answerPedro CrevillénJan 30, 2017AnswerYes, usually it is used a rotary vane pumps. But I learn that maybe a stroger vacuum is better (about 2 x 10-3 mbar / 1.5 x 10-3 Torr)Thanks.View0 Recommendations Bismark Oliver Cordero Lemanaasked a question related to Fluorescence In Situ HybridizationAside from using FISH, can someone suggest a differential stain for oomycete and true fungi?Question13 answersJan 26, 2017I am working with pathogenic oomycetes in grasses which are asymptomatic and rarely produce sporangia growing out from the host tissue. The grass samples were fixed in 5% Glutaraldehyde in PBS (pH 7.5) and thus difficult to proceed with oomycete-specific probes for FISH (GA creates a very strong fluorescent background that even my negative controls reacts with the different filters). Apparently, I tried using multiple fluorescent stains and combinations (Aniline Blue, Trypan Blue, and Uvitex2b) although these stains also binds to chitin. The problem is the host plant may have both fungal and oomycete pathogens together. Likewise, it is already sure that my target parasite is present since specific primers were designed (sequences obtained were 99-100% identical and forms a clade with the same species with 95% support by RaxML) and the samples for microscopy where derived from the same batch. I had also considered morphological aspects like the presence and absence of septation, but reports of septation in the hyphae of oomycetes had been widely observed in different genera (Plasmopara, Peronospora, etc). Trying chitin degradation had been an option too, but the procedure is too tedious, likewise the chances of degrading cellulose and glucans of oomycete cell walls are highly possible. Considering my little amount of samples, this risk is also quite the least option for me. Sorry for the long detail, I hope someone can offer some suggestions. Thanks!Relevant answerBismark Oliver Cordero LemanaJan 30, 2017Answerohhh. Thanks a lot. sorry for my delayed response. But these are really helpful. I will check them! Thanks againView0 Recommendations Sanda Raicasked a question related to Fluorescence In Situ HybridizationSuggestions how to increase mitotic index from primary fibroblast culture?Question7 answersJan 26, 2017I am trying to do fluorescence in situ hybridization(FISH) on primary culture of fibroblast. I was working with 25cm^2 flasks. Around 80% confluence, I treated cells with colcemid for 4H and followed regular protocol for chromosome harvesting of adherent cells. After I died cells with DAPI just to make sure that I did everything correct. However, I got high number of interphase cells and very few mitotic cells (1-2 per slide). Can anyone tell me what I did wrong and how to increase my mitotic index?Relevant answerPhilippe PoindronJan 28, 2017AnswerWhich concentration of serum do you use ? Try to decrease the number of cell seeded in order to avoid contact inhibition (even if you  treat cells before they reach confluence)and to boost cell proliferation. I disagree with your protocol of serum starvation. Serum is undispensable for fibroblast multiplication. (See papers of HAM et al. on cell nutrition types). In my opinion, your problem is linked to the absence of serum.View0 Recommendations maria cecilia Tacchiasked a question related to Fluorescence In Situ HybridizationProcessing time for cytogenetics in McCoy medium?Question2 answersJan 13, 2017Hi, anybody knows how fast a bone marrow sample for Fish or conventional cytogenetics taken from bone marrow and placed on McCoy medium needs to be processed?I work 5 hours away from the Research facility that does our cytogenetics and the admin personnel tell me it needs to be at the facility the morning after it was taken.Is that so? even in a culture medium? can there it be an interval of 36 hours between taking the sample an it reaching the facility?thanks!Relevant answerFatma Abdel WahabJan 20, 2017AnswerI agree with Sonal opinionView0 Recommendations Ruth Montalbo Calafellasked a question related to Fluorescence In Situ HybridizationAny idea to isolate DNA from cells used for FISH analysis fixed in a microscope slide?Question3 answersJan 19, 2017Before FISH analysis, cells are fixed in methanol:acetic acidRelevant answerSuzana RadojkovicJan 20, 2017Answertechnically yes as they wrote before, just take care, your DNA might be dirty or in insufficient amount, good luck View0 Recommendations Umar Burkiasked a question related to Fluorescence In Situ HybridizationHow to remove satellite cells from isolated mouse muscle fibres?Question4 answersJan 10, 2017I need to quantify the uptake of our therapeutic oligo drugs in the nuclei of muscle fibres isolated from adult mice. However these fibres have several satellite cells attached to them (see photo of an FDB fibre isolated from a 15wk old BL10 mouse) and therefore I need to remove them first in order to be certain that the oligo uptake measured in the nuclear fraction is from the muscle fibre nuclei only. Is there a way to remove or encourage migration of satellite cells quickly from the muscle fibres i.e. agitation, centrifugation etc?  FBD muscle fibre from BL10 mouse.jpg48.87 KBRelevant answerGary R. WalkerJan 11, 2017AnswerI agree with Denise. In my case It has been about twenty years since I isolated satellite cells to grow but as I remember it involved digestion and d damage to the fiber (myocytes). Can you use fluorescent or immune fluorescence microscopic approach to both distinguishing the satellite cells from the myocytes and also for measuring drug uptake?You may consider using myogenic stem cells and creating in culture myofibers (myotubes). I have been inducing myogenesis in C2C12 mouse myoblasts quite some time, it is easy.View9 Recommendations Perumal Arumugam Desinguasked a question related to Fluorescence In Situ HybridizationWhat is the ideal way of reconstituting an oligoprobe for fluorescent in situ hybridisation?Question4 answersJun 11, 2015We obtained a Fluorescent end labelled oligoprobe  for in situ hybridisation from a commercial source to detect viral nucleic acid. To prepare a stock solution from this newly synthesized probe what diluent is required? Can we use nuclease free water or any buffer is required?Relevant answerDebabrata PanjaDec 29, 2016AnswerHello,What is the commercial source for these oligo probes and did they work. Thanks View0 Recommendations Erman Salih Istifliasked a question related to Fluorescence In Situ HybridizationPepsin in fluorescent in situ hybridization?Question9 answersDec 9, 2016Hi everybody,I plan to perform FISH (telomeric and sentromeric probes at the same time) in human peripheral blood lymphocytes. Do you recommend using pepsin (is it compulsory??) or can I perform without using pepsin?Thank you very muchRelevant answerMaxim Yu. SinitskyDec 12, 2016AnswerDear Erman Salih Istifli,Pepsin is the very important component because it s a protease and acts to digest proteins into peptides and it helps to digest away cytoplasm on the slide. Digestion with pepsin can significantly improve probe penetration. Pepsin treatment is often useful when specimen preparations are suboptimal,e.g., in many clinical samples.Best regards,MaximView8 Recommendations Erman Salih Istifliasked a question related to Fluorescence In Situ HybridizationFluorescent in situ hybridization experiments with PNA probes?Question2 answersNov 3, 2016Dear AllIs it necessary to use RNase A  in fluorescent in situ hybridization experiments with PNA probes? Could FISH be performed without RNase A?Thank you very muchRelevant answerElodie LaharanneNov 3, 2016AnswerHi,No it s not necessary. See below. Which PNA do you use and what is your application?Protocole AppliedBiosystems.pdf101 KBView2 Recommendations Swojani Shresthaasked a question related to Fluorescence In Situ HybridizationCan anyone suggest a good Ki67 PI staining protocol for adherent cells?Question5 answersSep 26, 2016I used company s protocol for PI staining of human proximal tubule cell line. I fixed the cells using 70% EtOH and let them sit overnight at -20C, the next day followed staining protocol but the cell cycle histogram does not show a peak at G2M phase or there is a small scattered peaks that look similar to the S phase peaks. I have enough cell around 10^6 and I used 10ul PI for 100ul cell suspension. Could anyone suggest a good protocol where I can see distinct peaks for cell cycle phases. Also could someone elaborate the right setting on the cytometer for the PI detection like selecting A, W , H etc for appropriate analysis. Thank youRelevant answerDavid M SherrySep 28, 2016Answer      I wouldn t necessarily expect to see big, distinct peaks for G2/M or S. Most cells don t like to sit in G2 or M for very long. These phases can be pretty short, so you might not have a lot of cells in those phases at any given time. Do you synchronize the cells in your cultures so that they are all progressing through the cell cycle more-or-less together? This might yield tighter peaks.   If you need to visualize cells in different phases of the cell cycle in the microscope, the following might be useful.  We ve done experiments on cultured smooth muscle and fibroblast cell lines using Ki67 immunolabeling as a marker for cells in the cell cycle in combination with other cell cycle markers (cyclins) and DAPI to visualize nuclei and chromosomes for visualization in the microscope. The Ki67 and cyclin staining worked beautifully using fixation with 4% paraformaldehyde in 0.1 M PBS. (PI labeling also works under these conditions, although that isn t what we used for these experiments). EtOH fixation typically doesn t produce as a good a result as PFA does and can extract a lot of stuff from your cells. I would recommend EtOH fixation only if PFA doesn t work.  We had very good luck with a couple of different Ki67 antibodies from Millipore and from Cell Signalling and a cyclin antibody kit from Cell Signalling using PFA fixed cells.   Another approach that can be used in the microscope is EdU labeling (EdU is a BrDU analogue that gets incorporated into the DNA when it is replicated in S Phase). Molecular Probes has some very good fluorescent EdU kits.View6 Recommendations Qi Xiaoasked a question related to Fluorescence In Situ HybridizationHow to map the gene fusion by ISH?Question4 answersSep 24, 2016I m now trying hard to map the specific localization of a certain fusion gene by In situ hybridization (ISH), but I don t know how long the probe should I take in detection of pathological tissue and what is the proper method of setting corresponding control groups. If, on the other hand, the specific signals were to be detected, what should I do to exclude the possibility that signals are come from the separate genes?Relevant answerSaleh AlkarimSep 24, 2016AnswerHelloSee link and attachment here will help youGood Luck http://nopr.niscair.res.in/bitstream/123456789/5742/1/IJBT%204(3)%20307-315.pdfGeneral Introduction to in situ hybridization.pdf7.03 MBView3 Recommendations Hareschandra Jayaneththiasked a question related to Fluorescence In Situ HybridizationAny idea with this species?Question11 answersSep 12, 2016this eel observed in lagoon in western Sri Lanka. longer than 3.5 feet if you have any idea please help to identify  DSC00721.JPG1.97 MBDSC00824.JPG3.06 MBDSC00836.JPG3.19 MBRelevant answerRonald FrickeSep 14, 2016AnswerI agree with Philippe as well; the Giant estuarine moray, Strophidon sathete (Hamilton 1822) (family MURAENIDAE) matches with having a small head and a very long tail. The species lives in burrows in mud bottom, usually in estuaries and river mouths, in the Indo-West Pacific from the Red Sea and East Africa east to the Philippines and New Guinea.View12 Recommendations Victor Oforiasked a question related to Fluorescence In Situ HybridizationPlease how many years is the fluorescent in situ hybridisation technique effective for methanol-fixed bone marrow slides?Question2 answersSep 1, 2016I have archived slides which I want to detect cytogenetic abnormalities by FISH and would like to know the number of years of storage for which the slides will still be ideal for use.Relevant answerAnkita PatelSep 2, 2016AnswerThat is correct. I have been able to FISH on slides that were 2 years old- but they were stored at -20 with drierite chips to reduce humidity. If you have spare slides you may want to try the following that I have used on nuclei extracted from paraffin blocks that were quite old. Treat the slide with 50%glycerol in 2XSSC at 90C for 5-10 min. Dehydrate the slides in thorough ethanol series of washes- 70%, 80% and 100%. Look at the morphology of the cells - in a phase scope to see that they are still intact. I had to vary the treatment time depending on how old the blocks were. Then use your standard FISH protocol.View3 Recommendations Smita Kujurasked a question related to Fluorescence In Situ HybridizationWhy do we use 3% Igepal in FISH cytogenetics?Question5 answersAug 22, 2016We use wash solution on the 2nd day of FISH experiment. It contains SSC + 3% Igepal , a non-denaturing detergent. Can you specify what is it s action on the slides?Relevant answerDeepika GsSep 1, 2016AnswerDear Smita,In 1st day FISH ,Its function is same as said by Mr Pavel .But in 2nd day FISH processing,we use Igepal /NP-40/tween-20 along with 2X SSC or 0.4XSSC to remove the unbound probe.The concentration of SSC and detergent detemines the stringency  of washes.If concentration varies you might get false signals or non specific signals.View4 Recommendations David Wuasked a question related to Fluorescence In Situ HybridizationFluorescence in Situ Hybridization (FISH) Question: I am seeing both sense and anti-sense probe hybridization when FISHing. Any advice?Question1 answerAug 10, 2016I m using DIG-labelled probes and TSA-Cy3 from PerkinElmerRelevant answerSimone SchindlerAug 11, 2016AnswerHi David,There are a few things I could think of, that might cause staining (specific or unspecific) in your control.Do you see the same expression pattern for both the sense and the antisense probe? It is possible, that the reverse strand of your gene of interest encodes another gene, hence would be detected by your sense probe.Are you quenching the endogenous peroxidase, before you perform the staining? If not, that might be the reason for background staining.Are you blocking the tissue with blocking buffer, before incubating with the DIG antibody? If not, that might lead to unspecific binding of the antibody.I hope this helps.BestSimoneView0 Recommendations Mackenzie Hepkerasked a question related to Fluorescence In Situ HybridizationTrouble optimizing Oligo(dt)45 probe ISH in human brain tissue. Anyone have experience with this probe?Question2 answersAug 9, 2016New to this technique so sorry if this is poorly articulated, but here s the gist! We ve been having some trouble with an Oligo(dt)45 probe from Integrated DNA Technologies. The probe has been used successfully in several other studies found in the literature, on mouse/rat tissue--we haven t found any instances of application to human tissue yet.The probe is an HPLC purified DNA probe conjugated with 5 AlexaFluor 546, with the following sequence: 5 /5Alex546N/TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT 3 . We use in situ hybridization with the ultimate aim of visualizing mRNA. The Tm is 53.9*C, and we ve tried hybridizing the probe at temperatures of 44*C, 56*C, and 39*C for ~24 hours each with a standard protocol for paraffin-embedded, free-floating and fresh frozen (then short- or long-fixed) tissue. Nothing shows up under the microscope under any of the conditions we ve tried.A positive control (a sense foci probe) worked fine, indicating that it wasn t a technical issue. We checked out a microliter of the probe itself under the microscope, and it fluoresced beautifully on its own.We suspect the probe isn t binding for some reason and have contacted the manufacturer. Any tips for what other conditions (besides hybridizing temperature) we could play with, ideas about where we might be losing the probe, or tales of success would be greatly appreciated! Thanks.Relevant answerKaren NygardAug 11, 2016AnswerIf this is the first time establishing this in your lab, expect to have some troubleshooting to do. In situ hybridization is time consuming to troubleshoot as there are so  many steps where things can go wrong.It may be possible, especially with human-derived tissues, that losses of RNA have happened due to delays in fixation causing some necrotic change, over- fixation, or RNAse contamination in transit. We have more control in animal studies. The positive control may just be so much more abundant that you get some signal but your test signal is below the threshold.So, what to try? Here s a few starter ideas:SALTS:Vary the concentrations in the washes. Use a little more salt and you may get more background, but also more signal. Once you have a signal you can work on clean up by titrating down the salts.TEMPERATURE:Lower temperatures (seems you ve tried this). Try adjusting prehy, hyb and wash temperatures.PRETREATMENTS:Experiment with heat retrieval, Triton X-100, and proteinase K time/temperatures after deparaffinization to get the probes accessing their target. This can vary, and human derived pathology samples are often over-fixed and need more robust pretreatments to break the heavy cross links and get the tagged probe in there.A NOTE ON PROTEINASE K: each brand uses a different number of IU/mg. The units of activity are what matter, not the mg/ml. Thus if you are copying a paper based on mg/ml concentration, your brand may be less active and thus need more time ,hotter temp or a higher concentration. HYBRIDIZATION BUFFERS:A) Formamide-poorly deioinized formamide is a number one cause of poor hybridization in my experience. Even though it claims to be pre-deionized we always re-deionized it just before use in the hyb buffer. We make small batches and didn t store it long. ALSO-different GC contents of probes may need less/more percentage of formamide in the hyb buffer for success. We always used 50% formamide but some people claim success with varying this.B) Salmon sperm DNA or other nucleic acid blocker-must be FRESHLY denatured and quenched on ice the day of use.ENHANCEMENT:You could try Tyramide signal amplification if your signals are weak.RNASE HYGIENE:Make sure any washes touching the sample AFTER deparaffinization and before hybridization are totally RNASE free. DEPC treated buffers, clean pipettors and designated glassware that has been RNASE treated or baked at 450C overnight are mandatory at this stage. don t let anyone handle bacteria or RNASes near your bench.I hope this gives you some ideas about where to start. GOOD LUCK!View18 Recommendations Nikola Capkováasked a question related to Fluorescence In Situ HybridizationAny help/advice with replacement of PALM/dSTORM labelling dyes with STED Abberior dyes?Question2 answersAug 3, 2016I am working with PAML/dSTORM to visualize human mitochondrial DNA nucleoids. I try to visualize sequences of mtDNA by FISH, mostly with Alexa 647 and Cy3b. Now I have to find match for theese dyes from Abberior to be able use it for STED microscopy (to compare).Also, I am curious about theese two types: NHS activated and maleimid activated. Which one could be better for me? Thank you so much for help! http://www.sigmaaldrich.com/life-science/cell-biology/detection/abberior-dyes.htmlRelevant answerNikola CapkováAug 9, 2016AnswerThanks a lot! View0 Recommendations Smita Palasked a question related to Fluorescence In Situ HybridizationWhat exact color should be observed by CY3 tagged probe in FISH microscopy?Question3 answersJul 29, 2016Hi,I m trying to learn bacterial biofilm FISH microscopy.I used universal RNA probe EUB338c 16s tagged with Cy3. What exact color should be observed by CY3 tagged probe? I found my sample fluoresce dark pink, even many a paper suggested it must be red or pink, but I found its emission wavelength 570nm, that must be yellow zone. Is my result giving pseudochrome or there is any problem with my protocol?Relevant answerBiswaranjan PradhanJul 29, 2016AnswerThere is no problem with your protocol. I was also surprised when I first encountered this problem. The color of Cy3 is pinkish red and that of Cy5 is blue to the naked eye. However emission spectrum of Cy3 is in the greenish yellow and that of Cy5 is in the Red region. This happens if they were excited with monochromatic light equal to their  excitation maxima (Which is true when you are scanning it in a microarray scanner). But In Confocal/ FISH/ Floroscence microscope , the excitation spectrum contains a range of wavelengths which leads to a range in the emission spectra. That is why the color of Cy3 and Cy5 is same both in microscope and as you see in visible light. View8 Recommendations Tobias Vonnahmeasked a question related to Fluorescence In Situ HybridizationDoes anyone know where to get cultures of Nitrosococcus, wb1-A12, Nitrospira, or Nitrospina?Question4 answersJun 29, 2016For testing several FISH probes it would be very useful to work with cultures of these organisms (especially for Nitrosococcus and wb1-A12). It would be best to get these cultures from Europe, and even better if they were actively growing.Relevant answerTina SandersJun 30, 2016AnswerThe problem auf the nitrifier culture is that they normally not storied in instititions like the DSMZ, because the growing after freezing often not works. So you need active growing cultures.The microbiology of the University of Hamburg have a unique Stammsammlung of nitrifiers. Please ask Andreas Pommerening for the Ammonia oxidizers or Eva Spieck for Nitrite oxidizers.Cheers,TinaView16 Recommendations Katerina Pantopikouasked a question related to Fluorescence In Situ HybridizationF.I.S.H probe: Can be used as a probe a pcr labeled product with 600-1000 bps size?Question1 answerJun 6, 2016Is it nessessary to be inserted in a BAC clone?Relevant answerKatya MokretarJun 16, 2016AnswerYou can use a PCR product floursecntly labeled, the problem is if ti is not for repeated region and you have just one copy of it in the genome that you are not going to be able to visualise it. If you have a huge amount of repetitions there then there is no problem. It docent have to be in the BAC even better if it is not. The role of the BACs is just to amplify it with less chance of mismatching of nucleotides and introducing errors, but if you have it already  don t worry about the BACs.Hope this helpsKatyaView0 Recommendations Michał Bacaasked a question related to Fluorescence In Situ HybridizationDo probes for identification of E. coli i Pseudomonas fluorescens in FISH method have this sequences of nucleotides?Question1 answerJun 6, 2016The sequences are CCCCCTCTTTGGTCTTGC CCTTCCTCCCAACTT?Relevant answerHoward JuncaJun 8, 2016AnswerDetails for your question in:http://onlinelibrary.wiley.com/store/10.1111/j.1365-2672.2005.02800.x/asset/j.1365-2672.2005.02800.x.pdf;jsessionid=7E81C38F0099FC75063D31B1376CE0BB.f03t02?v=1 t=ip7bmni5 s=0665a8b88709262c13233b3541c6697ffcd32af4http://nanosims.univie.ac.at/fileadmin/user_upload/proj_nanosims/Huang_2007_Env_Micro.pdfView0 Recommendations Lixin Wangasked a question related to Fluorescence In Situ HybridizationWhat are the best retrograde or anterograde tracers for innervation to the intestine?Question2 answersMar 15, 2016We are trying to label 2 or 3 regions of the intestine at the same time by using different fluorescent conjugates. Since the neuronal elements in the gut are more sparse and injection areas much bigger than in the brain and spinal cord, we need more sensitive tracers which can be uptaken efficiently and travel long distance. Also, the tracers with different colored conjugates can label the neurons or terminals with the same efficiency. Relevant answerLixin WangJun 6, 2016AnswerMany thanks to Dr. Hoffman!View0 Recommendations Saurajyoti Karasked a question related to Fluorescence In Situ HybridizationMethods for Image segmentation of FISH microscopy images?Question6 answersMay 20, 2016Hi, I am trying to process FISH molecular biology experiment - microscopy images for quantification of fluorescence biomass. I have until now used some methods, but haven t got reliable results. For those who do such experiments, and those who work on image segmentation, I would like your suggestions which I can follow up.My main challenges are in background correction and automatic threshold calculation.In the link below, please find a sample set of 30 FISH images.https://www.dropbox.com/sh/a78yeetmfik72i7/AAAmCGalsmrJunUhdhYYoxU7a?dl=0Relevant answerSaurajyoti KarMay 25, 2016AnswerHello Narender, thanks for your mentions of the two articles. I shall go through them.Hello Elodie, yes indeed the images look very saturated. We tried to un-cluster the biomass but still a lot remains together. The experimentation is about detection of Nitrifiers in samples from treatment tanks of a waste water treatment plant. The magnification used is 40x which is used as standard in WWTP analysis as far as i know. Probe used is BET42a and general stain used is DAPI.Hello John, that shall be great. Yes, red and blue has to be separated. Am curious which software are you mentioning? Is it specifically developed for FISH? Is it a package as in use in Matlab or Java?Hello Norman, thanks for your suggestion. I shall look in the community you mentioned. The software you mentioned may be much complicated for the experiment we are doing And we aren t dealing with tissue but just biomass.Hello Christopher, i shall definitely try the CellProfiler and see how it works.View0 Recommendations Erica Molinaasked a question related to Fluorescence In Situ HybridizationAnyone know of a good protocol for fluorescent in situ hybridization using frozen tissue?Question3 answersMay 13, 2016I am looking for a good protocol for fluorescent in situ hybridization. I will be using flash frozen follicles from chickens treated with testosterone to look at the AR. The majority of the ones I have come across using different methods require a 2 week incubation period. Does anyone know of a good paper or have a protocol for this? Thank you!Relevant answerSimone SchindlerMay 14, 2016AnswerHi Erica,We have successfully used Fluorescent in situ hybridization for our study.http://dev.biologists.org/content/early/2016/04/26/dev.131292For our work we adapted the protocol from Jared Talbot for whole mount zebrafish larvae.https://wiki.zfin.org/display/prot/Triple+Fluorescent+In+SituFor more details on our protocol, you could visit the link below.I know, that this protocol is not specifically for the kind of tissue you are using, but, apart from the fixation and the permeabilization, all the following steps should be same.Please let me know, if you have questions.BestSimonehttps://insitutech.wordpress.com/2015/12/17/in-situ-hybridization/View4 Recommendations Joao Paulo Rodrigues Marquesasked a question related to Fluorescence In Situ HybridizationWhat is the function of triethanolamine during the in situ hybridization? How does this reagent interacts with the RNA?Question9 answersFeb 16, 2015I am doing in situ hybridization in plant tissues, but I have questions about this technique. What is the function of triethanolamine used in in situ hybridization. How does this reagent interacts with RNA? Can I change triethanolamine by other reagent?Thank you.Relevant answerJoao Paulo Rodrigues MarquesMay 4, 2016AnswerHi Piyush, Thanks for your comments. Well I was facing some problems to do my ISH last year. I verified that is necessary to use Triethanolamina and acetic anydre. I know that it s hard to get it, but you must use it! After I used those reagents my experients worked good, and then I got very nice results. Sure that you must have patient and do your sense and antisense probes to compare. But, if you spend some hours in the lab you able to do it. Please let me know any question concerning ISH. I am very interested and help and learn about this usefull tool.Regards,João PauloView16 Recommendations David Schwartzasked a question related to Fluorescence In Situ HybridizationCan someone give me advice on how to design FISH probes?Question2 answersApr 15, 2016Are there programs available to do this?Relevant answerThomas LiehrApr 23, 2016Answeryou may check for putative FISH-results of human probes at link providedhttp://projects.tcag.ca/cgi-bin/efish/index.cgiView0 Recommendations Sangeet Bhaumikasked a question related to Fluorescence In Situ HybridizationHow to normalize the fluorescent data obtained from spectrofluorometer?Question3 answersApr 15, 2016I have a target gene, which is differentially expressed in lung cancer patient, house keeping gene, and a third gene which is highly expressed in all lung cancer patients. I took the fluorescent signal for all the genes after hybridizing them with a particular fluorescent dye. Now I have the raw data for every gene and their concentration. I want to see the concentration difference of the target gene in a patient sample and normal sample. What statistical method should I choose and what data normalization procedure should I follow?Relevant answerTetyana BeltramoApr 19, 2016AnswerHello,there is a number of methods to pre-process your data. You should try all of them to find out the best one for your data set. For example, standard normal variate, multiplicative csatter correction, extended multiplicative scatter correction, generalized least square weigtning filtering.Best regardsView5 Recommendations Ronaldo Martins Júniorasked a question related to Fluorescence In Situ HybridizationWhat is a good DAPI concentration for FISH nuclear counterstaining?Question3 answersApr 6, 2016Hello all,I have a stock of DAPI at 1mg/ml and I want to know the best concentration for FISH nuclear counterstaining.As well, what is a good solvent to disolve it in? It is a good idea disolvethe DAPI in anti-fade reagent?Any and all suggestions are welcome.Relevant answerJudith ZimmermannApr 10, 2016AnswerIn our lab we commonly stain cells/ tissue for 5- 10 min in 1 µg/ ml DAPI solution (in RNAse-free water) and after washing, embedd the cells/ tissue in a 11:2 mix of Citifluor (Citifluor Ltd, London, U.K) and Vecta Shield (anti bleaching agent) (Vector Laboratories, Inc., Burlingame, CA).Others dilute DAPI directly in the Citifluor/Vectashield mix (1- 2 µg/ ml and use that as embedding mix. This works for example really nicely if you experience problems, such as fast bleaching of the DAPI signal (varies for different cells/ tissues). To make sure nothing of the DAPI is spilled, we commonly use nail polish to seal the slides before microscopy.Hope that helps!View3 Recommendations Thomas Jansasked a question related to Fluorescence In Situ HybridizationWhat is the use of MgCl2 and EtOH washes in RNA FISH?Question1 answerApr 6, 2016We are doing RNA FISH, we have this new protocol that does not have any MgCl2 and EtOH washes of the samples, something we usually do.I was wondering what the use of these steps was.I ve found that MgCl2 might be used for pepsin deactivation in case of pepsin pre-treatment (which we don t do) and that EtOH keeps the RNA single stranded, but cannot confirm this.Relevant answerBeryl Schwarz-HerzkeApr 7, 2016AnswerDear Thomas,as I know is EtOH added to avoid hydration of nucleic acids so that the binding of the probes is not inhibited. This is the same while precipitation on RNA or DNA. But to keep RNA single stranded, EtOH is not really helpful (as I know).MgCl2 ios often used to stabilize the cytosceleton. It is fixing g and F proteins and other cytoskeleton elements. But for Fish, you want to permeabilize the cell membrane, so I think ypo can avoid it. I never did this proceedure using MgCl2 and it worked.View12 Recommendations Jaime de Juan-Sanzasked a question related to Fluorescence In Situ HybridizationCan one use RNA-FISH to detect shRNA efficiency in single cell?Question7 answersSep 18, 2015The idea is to transfect shRNA vector sparsely in neurons and check its efficiency by RNA-FISH individually in each of the transfected cells. I believe it should be possible but I can t find any publication doing this.Relevant answerJaime de Juan-SanzMar 31, 2016AnswerHi Hans,Indeed, it can be done. We solved it some time ago. Thanks for your answer, though. Best,JaimeView0 Recommendations Samatha Mathewasked a question related to Fluorescence In Situ HybridizationHow to tackle high background signal in RNA- fluorescence in situ hybridisation (FISH)?Question5 answersJan 26, 2016Hi,In RNA- FISH using biotinylated probes and streptavidin- Cy3, I get a high background signal, even in the no probe control. I am using 5% BSA for blocking prior to incubation with SA- Cy3, and give subsequent washes with 2X SSC and PBS. How can I fix the problem? Thanks.Relevant answerHans E. JohanssonMar 31, 2016AnswerNot trying to sell anything here, but have you tried Stellaris RNA FISH probes?Compatible with GFP.HansView0 Recommendations Alessandra Obertoasked a question related to Fluorescence In Situ HybridizationHas anybody used 45base oligos to do in situ hybridization of mRNA?Question1 answerMar 29, 2016I would like to use 45mers oligonucleotides (3 or 4) for in situ hybridization of mRNA of a very low expressed gene. I have to use DIG labelled probe. I would like to try to order the oligo rady with  aDIG tail. but at Sigma they said that thay have not idea how long should be the DIG-tail. Please help me if you have any idea about using oligo for DIG- in situ hybridization or related to non radioactive in situ. Thank you. AlessandraRelevant answerAlessandra Maria Adelaide ChiottoMar 30, 2016AnswerSalve Prof. Io no, ma il gruppo di Giorgio Merlo fa Ish, non so con che tipo di oligo però può provare a chiedere a lui. ☺View0 Recommendations Muhammad Arslanasked a question related to Fluorescence In Situ HybridizationCan anyone suggest some appropriate methods to preserve plant tissues for future endophytes studies?Question3 answersMar 11, 2016Hi All,I am looking for opinions to preserve plant tissues for endophytes (bacteria living inside of plant tissues) analysis in near future. Primary objective is to do fluorescent in situ hybridization at later stages of my experiment. Moreover, do you think that cryopreservation techniques may work for this?Thanks in anticipationRelevant answerZakir HossainMar 15, 2016AnswerThis article (doi:10.1126/science.aaa8764) might be helpful although they did not use the method for in situ hybridization. They developed the method for endophytes isolation.View5 Recommendations Tamara Müllerasked a question related to Fluorescence In Situ HybridizationDo Ibidis (µ-slide 8 well) tolerate liquid nitrogen?Question4 answersMar 12, 2016I am performing on slide FISH and a part of the protocol consists of incubation in liquid nitrogen. Due to the fact that my cells don t grow on pre-treated glass slides I had to switch to Ibidi-slides. Does anyone have experience with the temperature stability of the Ibidis?Thanks in advance. Relevant answerTamara MüllerMar 14, 2016AnswerActually, I tried it this morning. First I dipped the slides 5x in LN2 and afterwards placed them on a hot plate (70°C) and they seem to be very fine.Thanks a lot, Venkatesh!View3 Recommendations Hadil Alahdalasked a question related to Fluorescence In Situ HybridizationHow I can combine ISH with IF using mouse brain tissue?Question2 answersFeb 29, 2016I am trying FISH on mouse brain tissue, but I got cross reactivity between the miR signal and the antibody I used.Brains are fixed with 4% PFA, cryopreserved and sectioned (12.5um thickness). I am using a DIG probe for a micro RNA, and the ISH for the selected probe is working. I want to localise the probe signal so I select a number of neuronal and astrocyte markers for that. However, when I did that I had cross-reactivity, despite using antibodies raised in different animals. I tried to use different blockings like 10% NGS or NDS and 1% BSA, still the same problem. Also, I changed the antibody, but with no help. When I stained the antibody alone, it worked!I am using 50 degrees for Hibredisation and 60 degrees for washing in SSC.Relevant answerHadil AlahdalMar 1, 2016AnswerThanks Yingjun Liu,View0 Recommendations Priyanka G Bhosaleasked a question related to Fluorescence In Situ HybridizationHow to manually enumerate FISH signals for gain versus amplification?Question3 answersFeb 24, 2016While enumerating FISH signal for a locus, I get wide range of signal per nuclei (i.e  6, 7, 9 or even 10 signals per nuclei). How should I categorize these signals for gain or amplification. Should I consider, more than 6 signals per nuclei as amplification or it will be a high copy gain.  Relevant answerAndreas H ScheelFeb 28, 2016AnswerHey, for diagnostic FISH of HER2/neu, MET and FGFR1, different cut-offs are used that have been validated in clinical trials, e.g. Clin Cancer Res. 2015 Feb 15;21(4):907-15. As a starting-point you could use several criterions and test if they separate your probes into meaningful groups, e.g.Copy-number: =4, 6 : low-level =6 : High levelRatio (Target probe / Reference probe): 2.0 : AmplifiedAre normal cells present that you might use as internal control?Do you see separated signals or clusters of signals in the cells of interest?Best regardsView12 Recommendations Boudiaf Anisasked a question related to Fluorescence In Situ HybridizationHER2 fluorescent in situ hybridization FISH DAKO kit ?Question3 answersFeb 20, 2016i m using DAKO kit for HER2 fluorescent in situ hybridization FISH and i would like to ask if it is possible to stop the procedure after hydridization and continue later with the protocol ?HER2 FISH on breast cancer formalion fixed and parafin samples with dako kit Relevant answerBoudiaf AnisFeb 21, 2016Answerameya, it s just due to some timing problems in the lab so sometimes i have to stop my experiment after hybridization View5 Recommendations Joon Choiasked a question related to Fluorescence In Situ HybridizationIs it possible to fix yeast cells in a flow chamber?Question1 answerFeb 12, 2016Is there any one who knows how to fix Saccharomyces cerevisiae in the microfluidics device (basically not in cell culture) and do RNA FISH? All protocols I have read fix cells in the culture. I am wondering how efficiently fixation can be done in the flow chamber. Please let me know if you have any experience about this. Thank you so much.Relevant answerInga Morkvenaite-VilkoncieneFeb 15, 2016AnswerHi, Joon,We use poly-L-lysine for cells fixation. But the strongest fixation could be achieved, if cells are entrapped in polymer mebranes.View0 Recommendations Anchit Khannaasked a question related to Fluorescence In Situ HybridizationHow much concordance is there between Immunohistochemistry (IHC) FISH testing for detecting mutation-dependent overexpression of target kinase ?Question2 answersFeb 6, 2016There is a good /validated antibody for doing the IHC.  Relevant answerAnchit KhannaFeb 11, 2016AnswerThanks Jukka !  Though I do wonder what FISH would bring to the table, especially for stratification of patients for targeted therapies. As you could possibly only detect a single variant  with a single set of FISH probes which would cost you 3-4 times an IHC.  Whereas in case of IHC you would  recognise  the end product (protein)  regardless of  the upstream rearranged variant. Ofcourse IHC is as good as the antibody and the  controls.View0 Recommendations Umar Burkiasked a question related to Fluorescence In Situ HybridizationHas anyone used Fluorescence in situ Hybridization (FISH) for detecting oligonucleotides in mouse tissues or cells?Question5 answersFeb 5, 2016I am trying to develop a Fluorescence in situ Hybridization (FISH) assay for visualising the sub-cellular localisation of 25-mer oligonucleotide drugs in mouse tissues. The aim is to show the distribution of the oligo after it’s taken up and how much enters the target nucleus compartment. My aim is to use the simplest method of FISH in the first instance, where I will use an Alex-488 labelled DNA probe complementary to the oligo. If that doesn’t work then I will consider using Dig-labelled DNA probe and subsequent Alex-488 anti-dig antibody. If someone has any experience of using FISH assays for Oligo detection then please get in touch. I would appreciate any tips and advices anyone can offer.    p.s. I am aware of sub-cellular fractionation kits for proteins which can be used for this purpose but I would like to give FISH assays a go first.Relevant answerTobias Aurelius KnochFeb 10, 2016Answerunspecific binding, steric trapping, or naturally the hybridication = so that is, of course, one thing you might need to consider if you did not see it...but if you want to see how oligos distribute as oligos, so not for FISH, then you should do this in vivo - send me an email, and let s get in contact, but you might consider the following papers for that:Counting nucleosomes in living cells with a combination of fluorescence correlation spectroscopy and confocal imaging. Weidemann T, Wachsmuth M, Knoch TA, Müller G, Waldeck W, Langowski J. J Mol Biol. 2003 Nov 21;334(2):229-40.Analyzing intracellular binding and diffusion with continuous fluorescence photobleaching. Wachsmuth M, Weidemann T, Müller G, Hoffmann-Rohrer UW, Knoch TA, Waldeck W, Langowski J. Biophys J. 2003 May;84(5):3353-63.Trichostatin A-induced histone acetylation causes decondensation of interphase chromatin. Tóth KF, Knoch TA, Wachsmuth M, Frank-Stöhr M, Stöhr M, Bacher CP, Müller G, Rippe K. J Cell Sci. 2004 Aug 15;117(Pt 18):4277-87. Epub 2004 Aug 3.Detection of NGF-receptors TrkA and p75NTR in human tumor cell lines and effect of NGF on the growth characteristic of the UT-7/EPO cell line. Westphal G, van den Berg-Stein S, Braun K, Knoch TA, Dümmerling M, Langowski J, Debus J, Friedrich E. J Exp Clin Cancer Res. 2002 Jun;21(2):255-67.so and for high resolution stuff and quantification:Light optical precision measurements of the active and inactive Prader-Willi syndrome imprinted regions in human cell nuclei. Rauch J, Knoch TA, Solovei I, Teller K, Stein S, Buiting K, Horsthemke B, Langowski J, Cremer T, Hausmann M, Cremer C. Differentiation. 2008 Jan;76(1):66-82. Epub 2007 Nov 26.The 3D structure of the immunoglobulin heavy-chain locus: implications for long-range genomic interactions. Jhunjhunwala S, van Zelm MC, Peak MM, Cutchin S, Riblet R, van Dongen JJ, Grosveld FG, Knoch TA, Murre C. Cell. 2008 Apr 18;133(2):265-79. doi: 10.1016/j.cell.2008.03.024.Super-resolution imaging reveals three-dimensional folding dynamics of the β-globin locus upon gene activation. van de Corput MP, de Boer E, Knoch TA, van Cappellen WA, Quintanilla A, Ferrand L, Grosveld FG. J Cell Sci. 2012 Oct 1;125(Pt 19):4630-9. doi: 10.1242/jcs.108522. Epub 2012 Jul 5.View0 Recommendations Kunal Chopraasked a question related to Fluorescence In Situ HybridizationHas anyone tried an in situ in adult zebrafish tissues?Question7 answersJan 18, 2016I am planning to do an in situ to determine if a gene of interest is expressed in the intestine. I have done in situs in larval zebrafish but never in adults/adult tissue. Can anyone point me towards any key technical papers where I can make a start? Thank youRelevant answerSimone SchindlerJan 20, 2016AnswerHi Kunal,I have done Fluorescent RNA in situs on paraffin sections for adult zebrafish heads. Below is the link for the blog I wrote about it. https://insitutech.wordpress.com/2015/12/17/in-situ-hybridization/Maybe this could help. Let me know, if you have questions. Best SimoneView21 Recommendations Anissa Niniasked a question related to Fluorescence In Situ HybridizationHow can I do Fluorescence In Situ Hybridization on Formalin-fixed paraffin-emebedded tissue sections ??Question5 answersJan 14, 2016procedure pleaseRelevant answerSimone SchindlerJan 14, 2016AnswerHi Anissa,I am assuming you want to detect RNA. Attached is the link for the  protocol I used for performing fluorescent in situ hybridization on zebrafish and mouse paraffin sections.https://insitutech.wordpress.com/2015/12/17/in-situ-hybridization/I hope this helps. Good luck.SimoneView2 Recommendations123AdvertisementJoin ResearchGate to find the people and research you need to help your work.20+ million members135+ million publications700k+ research projectsJoin for freeSimilar topicsCytogeneticsIn Situ HybridizationImmunohistochemistryPCRImmunohistochemical StainingImmunofluorescence MicroscopyImmunofluorescence StainingMicroscopyFluorescence MicroscopyHistological TechniquesHighly-cited researchers Faisal KlufahAlbaha UniversityDidn t find what you re looking for?Search for more research, methods, and experts in other areas on ResearchGate.Discover more researchAdvertisement orDiscover by subject areaRecruit researchersJoin for freeLoginEmail Tip: Most researchers use their institutional email address as their ResearchGate loginPasswordForgot password? Keep me logged inLog inorContinue with GoogleWelcome back! 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