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Wholemount in situ hybridisation

来自 : 蚂蚁淘

Wholemountinsituhybridisation

BasedonWilkinsonprotocol,modifiedbyMurrayHargrave(m.hargrave@cmcb.uq.edu.au),Koopmanlab

A.SYNTHESISOFPROBE

1Mixinthefollowingorderatroomtemperature:

steriledistilledwater9.5µL
5xtranscriptionbuffer4µL
0.2MDTT1µL
nucleotidemix(pH8.0)2µL
(10xDIGRNAlabellingmix)
linearisedplasmid(1µg/µL)1µL
placentalribonucleaseinhibitor(40U/µL)1.5µL
Sp6,T7orT3RNApolymerase(20U/µL)1µL

2Incubateat37°Cfor1hour,thenaddanother20UofRNApolymerase.

3Incubateforafurtherhourat37°C.

4Removea1µLaliquotandrunona1%agarose/TAEgeltoestimatetheamountsynthesised.AnRNAbandofapproximately10foldgreaterintensitythantheplasmidbandindicatesthat~10µgofprobehasbeensynthesised.

5TherearetwosuccessfulmethodsforpurificationoftheRNAprobe:

A)forthelazy(thisworksfine):

Dilutetheprobeto50µlwithDEPCmilliQH2O,add5µlofRNasefree3MNaOAc,mixandadd2.5volumesofRNasefreeabs.ethanol.
Incubateat-20°Cfor30minutestopreciptatetheRNAandspindowninarefrigeratedmicrofugefor10minutes.
Washthepelletwell(twice)withRNasefree70%ethanoltogetridofanyunincorporatednucleotides

B)fortheparanoid:

Add2µLofRNasefreeDNase1(10U/?l).Incubateat37°Cfor15minutes.
PackaSephadexG50columnequilibratedwith0.1%SDS,50mMTris.Cl(pH7.5),0.5mMEDTA(TES)byloADInga1mlsyringebarrel(pluggedwithglassorplasticwool)andspinning@1500rpm90sec.
Diluteprobereactionto100?lwithTESandloadontopackedG50column.Collectfractionandloadanother100?ltothecolumnandspinagain.
Poolthefractionsandadd1/10volumeNaOAc,2volumes100%EtOHandincubateat-20°Cfor30minutes.
Spininarefrigeratedmicrofugefor10minutes,washpelletwithice-cold70%EtOH
Airdrypellet

6RedissolvepelletinDEPCmilliQH2Oat~0.1µg/µLandstoreat-20°C.

B.PRETREATMENTOFEMBRYOS

Allstepsarecarriedoutin4mLround-bottompolycarbonatesnap-captubesunlessotherwisestated.Enoughliquidmustbeusedtoensurethatallembryosarecompletelycoveredduringeachstep.
Allstepsarecarriedoutwithsufficientrockingtoagitate(butnotdestroy)theembryos,andunlessotherwisestated,atroomtemperature.
AllstepsuptoandincludinghybridisationarecarriedoutinRNase-freeconditions,usingRNase-freesolutions,wearingglovesetc.
Forthewashesat55°Cor65°C,itisconvenienttouseaheaterblockplacedonitssideonarockingplatformorahybridizationovenwithrotatingcylinders.

Day0

1Dissectembryosinice-coldPBS.TrytoremoveasmuchoftheextraembryonicmembranesaspossIBLe.Besuretoremoveoratleastpuncturetheamnionandinembryosof10dpcorolder,puncturetheheadwithasyringeneedletoavoidtrappingofreagentsinthelumen.

2Fixin4%paraformaldehyde(PFA)inPBSat4°Covernight.

Varyingthefixationtimefrom3htoovernighthasnoeffectonsignalorbackground.

3WashtwicewithPBTXfor10minuteseachat4°C.

4Washwith50%,MeOH/PBTX,thentwicewith100%MeOHfor10minuteseach.

Canstoretheembyrosat4°Cor-20°Catthispoint,foruptoafewmonths,orpreferablyinpre-hyb(seestep12).

Day1

5RehydratebytakingtheembryosbackthroughaMeOH/PBTX(75%MeOH->50%MeOH->25%MeOH->PBTX)seriesinreverse

6WashtwicewithPBTXfor10minuteseach.

7Treatwith10µg/mLProteinaseKinPBTXfor5-20minutesatroomtemperature.

ThelengthofthistreatmentdependsonthesizeofthesampleandthebatchofproteinaseK.Eachbatchshouldideallybetested.Asaroughguide,use5minforE7.5,7minforE8.5,9minforE9.5,11minforE10.5,12-14minforE11.5.

8WashtwicewithPBTXfor5minuteseach.
Becareful-theembryosarefragile!
9Refixwithfresh0.2%glutaraldehyde/4%PFAinPBTXfor20minutes.
10WashtwicewithPBTXfor10minuteseach.

11Placeina2mlscrewcapEppendorftubeandfillwithprehybridisationmix,andallowtheembryostosink,replaceprehybsoln.

12Incubateat65°Cfor2h.

Thisstepcanalsobeperformedovernight.Alternativelytheembryoscanbestoredinthissolutionat-20°C.

13Removeprehybandaddhybridisationmixincluding1.0µg/mLDIGlabelledRNAprobe.
Ifhighbackgroundisseen,probeconcentrationcanbedecreasedto0.5µg/mL.
Thetubeneedstobefullsothatprobedoesnotdryontothesample.
14Incubateat65°Covernight.

Iftheprobeisshortorheterologous,55°Ccanbeusedforpre-hyb,hybandstringencywashes.

Day2

C.POST-HYBRIDISATIONWASHES
FromthispointonRNase-freeconditionsarenolongernecessary.
1Washwiththefollowingfor5minuteseachat65°C(or55°C)
100%Solution1
75%Solution1:25%2xSSC
50%Solution1:50%2xSSC
25%Solution1:75%2xSSC
Duringthesewashes,startpreabsorbingtheantibodyasdescribedbelow.
2Washwith2xSSC,0.1%CHAPStwicefor30minuteseachat65°C(or55°C).
3Washwith0.2xSSC,0.1%CHAPStwicefor30minuteseachat65°C(or55°C).
4WashwithTBTX,twicefor10minuteseachatroomtemperature.

5Preblocktheembryoswith10%sheepserum,2%BSAinTBTXfor2-3hoursatroomtemperature.

6Removethe10%sheepserum,2%BSAfromtheembryosandreplacewiththepreabsorbedantibody(seebelow).Rockovernightat4°C.

D.PREABSORPTIONOFANTIBODY

1Duringthewashingoftheembryos(step1above),weighout3mgofembryopowderintoamicrotube,add0.5mLof10%sheepserum,2%BSAinTBTXand1µLanti-DIG-APFabfragment(Boehringer1093274).

Embryopowdershouldmatchthespeciesbeingstudied.

2Rockgentlyat4°Cfor3hoursorlonger.
3Spininamicrofugefor10minutesat4°C.
4Dilutethesupernatantto2mLusing10%sheepserum,2%BSAinTBTX.
5Storeat4°Cuntiluse.

E.POST-ANTIBODYWASHESANDHISTOCHEMISTRY

Day3

1WashatleastfivetimeswithTBTXcontaining0.1%BSAfor1houreachatroomtemp.
Theantibodysolutioncanbekeptat4°Candreusedupto4times.
2Washovernightat4°CwithTBTXcontaining0.1%BSA.
Thiswashisoptionalbutusuallyconvenient.

Day4

3WashtwicewithTBTXfor15minuteseach.
4WashthreetimeswithNTMTfor10minuteseach.

5IncubatewithNTMTincluding4.5µLNBTand3.5µLBCIP(X-phosphate)permL.Rockforthefirst20minutesthentransfertheembryostoaglassembryodishorscintillationvial.

Avoidusingaplasticpetridishascrystalstendtoform.

Keepinthedarkasmuchaspossibleandallowthecolourreactiontoproceeduntilsignalisstrongestwithoutproducingbackgroundstaining.

Itisbesttoslightlyoverstain,assubsequentwashingswilltendtodestainsamples.Ifsamplesaretobesectioned,overstainingisrecommended.YoucanstopthecolourreactionbywashinginNTMT,thenTBTXovernight,thenre-startthecolourreactionthenextmorning.

6Whenthecolourhasdevelopedtothedesiredextent,washwithNTMTthenwithPBTX.

7WashseveraltimesinPBSwith1%TritonX-100.

Thiswillbluethestainanddecreasebackgroundandsignal.Someobservationandjudgementisrequiredhere.Forweaksignal,thisstepcanbeshortenedoromitted.Ifsignalisstrongandbackgroundisweak,thenatotalofafewhoursisrecommended.Overstainedorhighbackgroundsamplescanbewashedforuptoseveraldays.

8Fixthestainbyincubatingtheembryosin4%PFAinPBTXovernightat4°C.

9Photographembryosassoonaspossible.

Thesignalcanfadeortheenitreembryocanturnblueuponstorage.Positionembryos,immersedinPBS,ingroovescutinalayerofagaroseinapetridish.Adjustlightingtooptimizethetranslucencyofthesample.

10Iftheembryosaretobestoredforextendedperiods,usePBScontainingsodiumazide,ortakethemthroughaPBTX/glycerolseriesinto100%glycerol.

SOLUTIONS

10xtranscriptionbuffer(forprobepreparation):400mMTris.Cl(pH8.25),60mMMgCl2,20mMspermidine.ThisissuppliedwithPromegapolymerases.
Nucleotidemix:10mMGTP,10mMATP,10mMCTP,6.5mMUTP,3.5mMDIG-UTP.ThiscanbeboughtfromBoehringerMannheim.
4%PFAinPBS(pH7.4)-preparedaheadoftimebydissolvingthepowderat65°C,andthencooled.Thiscanbefrozeninaliquots.
25%glutaraldehyde:storeinaliquotamountsat-20°Candthawjustpriortouse.
PBTX:PBS(pH7.4)with0.1%TritonX-100
Prehybridisationmix:50%formamide,5xSSC,2%Boehringerblockingpowder(cat.no.1096176,dissolvedirectlyintothemix),0.1%TritonX-100,0.5%CHAPS(SigmaC-3023),50µg/mLyeastRNA(SigmaR-6625),5mMEDTA,50µg/mLheparin.Forhybridisation,addprobeto1µg/mL
Solution1:50%formamide,5xSSC,0.1%TritonX-100,0.5%CHAPS
TBTX:50mMTris.Cl(pH7.5),150mMNaCl,0.1%TritonX-100
Mouseembryopowder:Homogenise~12.5-14.5dpcmouseembryosinaminimumvolumeofPBS.Add4volumesofice-coldacetone,mixandincubateonicefor30minutes.Spinat10,000xgfor10minutesandremovesupernatant.Washthepelletwithice-coldacetoneandspinagain.Spreadthepelletoutandgrindintoafinepowderonasheetoffilterpaperandallowittoairdry.Storeinanair-tighttubeat4°C.
NTMT:100mMNaCl,100mMTris.Cl(pH9.5),50mMMgCl2,0.1%Tween-20.

ThismustbemadefreshonthedayofuseaspHdecreasesduringstorageduetotheabsorptionofCO2.

NBT:75mg/mLindimethylformamide(storeat-20°C)

BCIP:50mg/mLindimethylformamide(storeat-20°C)

Itisbesttoletthesereagentswarmtoroomtemperaturebeforeuseasitmaydecreasetheamountofcrystalformationinthecolourreaction.

REFERENCE

Wilkinson,D.G.(1992)Wholemountinsituhybridizationofvertebrateembryos,inInsituhybridization:APracticalApproach(D.G.Wilkinson,ed)pp75-83,IRLPress,Oxford.

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发布于： 2018-12-26 阅读（143）

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