Preparationoftheprobe.

• ProbesarepreparedasDigoxigeninlabelledRNA.ThelabellingmixaswellasallantibodiesarepurchasedfromBoehringer
• AllconditionsandsolutionsshouldbetotallyRNAsefree.
• Useglovesandaerosolbarriertips.

1. Linearizetheplasmidandcheckthedigest.
2. Phenolextract.
3. Extracttwicewithchloroform:isoamylalcohol(24:1)
4. Ethanolprecipitate(1/2vol7.5MNH4OAc+2.5vol100%ethanol.Rinsewith125µl75%ethanol.Letdrywithcapsopenfor10minutes.)
5. ResUSPendinsuitablevolumeofnucleasefreewater.
6. Measureconcentration.
7. Setuptranscriptionreaction
   • 200ngDNA
   • 2µl10Xtranscriptionbuffer(Stratagene)
   • 2µllabellingmix
   • 1µlRNAGUARD(Pharmacia)
   • 1µlRNApolymerase(SP6,T3orT7)
   • Addwaterto20µl
8. Incubatefor2hoursat37°C.
9. Runon1%agarosegel(1.5µlprobe+5µlof1.2Xrunningbuffer.)forashorttime.TheRNAshouldappearasasinglebandwithlittledegradationproductandabout10timesmoreintensethantheDNAband.
10. RemoveunincorporatedfreenucleotideswithQuick-SpinColumns.
    • Removethecaps(topfirstnottocreateairbubbletrappedinthecolumn)andspinfor5min,@4°C,1800rpmintheSorvalswingbucketcentrifuge.
    • Removetheeluateandcentrifuge5minagain.
    • Putthecolumninnewtubes,addthetranscriptionreactionontothemandspin15min.
    • Thevolumeofthefinaleluateshouldbearound30-40µl.
    • Runagel(loADIng1.5µlin5µlloadingbuffer)toquantifytheyieldandtodeterminetheamounttobeusedfortheinsitu.

GeneralComments.

• Don'tlettheembryosdryatanystageastheamountofbackgroundwillincrease.Itispreferedtoleavetheembryosinasmallvolumeofthesolutionandtoaddthenextsolutiontoit.
• TreatallsolutionswithDEPC(add0.1%DEPC,incubatewithagitationovernightandautoclave40minutes)(forTrissolution,useDEPCtreatedwater,donottreatthesolution).
• Filterallsolutions(toremoveparticlesthatwillsticktotheembryos).
• RinsethehybridizationvialsandcapswithRNAZapandrinseatleast5timeswithDEPCwater.
• Makeallthefixations,rinses,washesuntilthepre-hybridizationsteponiceexcepttheproteinaseKtreatment.
• Useglovesandaerosolbarriertipsforchangingthesolutionsfromthefixationsteptotheendofhybridization.

Preparationofembryos

1. DissectembryosincoldPBS,changesolutionoften.
2. Punchaholeinbraincavitiesforembryosolderthan9dpc.
3. Transferafterdissectingafewembryostoa5mlscrewcapflatbottomedglassvialcontaining4%paraformaldehyde(freshlymade.AddpowderparaformaldehydetoPBSandheatto60°Cwithstirringuntilclear)Storeonice.
4. Whenalltheembryosofthesamemotheraredissected,renewthe4%paraformaldehydeandincubateat4°Cfor4hrsfor7.5dembryosorovernightforolderembryos(oroverdayifdissectionisdoneinthemorning).
5. Thenextday,wash2xwithPBSw(PBSw=PBSwith0.1%Tween-20)
6. Dehydratewithmethanolseries(25%,50%,75%,100%inPBSw).Change2xin100%methanol.
7. Storetheembryosat-20°C(upto2months).

InSituhybridization

Day1.

1. Preparefresh4%paraformaldehyde-0.2%GlutaraldehydeinPBS.About5mlwillbeneededforeachsampleafterproteinaseKtreatment.
2. Preparehybridizationsolution.For50mlofhybridizationsolutiondissolve;

• Hybridizationmixrecipe(50ml):
  • 0.5gBoehringerBlock
  • 25mlformamide
  • 12.5ml20XSSC,pH7
  • Heatto65°Cforabout1hr.Oncedissolvedadd:
  • 6mlH2O
  • 5ml10mg/mltorulaRNA(heat2minat65Ctoclear)
  • 100µl50mg/mlheparin
  • 250µl20%Tween-20
  • 500µl10%CHAPS
  • 500µl0.5MEDTA
• Filterthesolution.Thehybridizationsolutioncanbepreparedbefore,aliquotedandstoredat-20°C.

1. Rehydratetheembryosthrough75,50and25%methanolseriesinPBSw.Incubateeachstepfor5min.onice.
2. Wash3timesfor5min.withPBSwonice.
3. Changeto1mlof4.5µg/mlProteinaseKinPBSw.Incubatefor3minfor6dpcatRT,5minfor7.5dpc,7minfor8.5dpc,9minfor9.5dpc,11minfor10.5dpc,13minfor11.5dpc.Stainingforhighlyexpressedgenerequireslessdigestion,butforlowexpressiongeneslongerdigestionmayhelptogetstrongerstaining.MakesuretothawtheproteinaseKstockcompletelyandvortextodissolveprecipitateatthebottomofthetube.UsealiquotsoftheproteinaseKstock10mg/ml,donotthaw-freezerepeatedly.(Incubationtimeshavetobeoptimizedforeachstock.)
4. Stopdigestionbywashinginfreshlyprepared2mg/mlglycineinPBSW
5. RinseinPBSw.
6. Wash2timeswithPBSwfor5min.
7. Refixin5mlof4%paraformaldehyde-0.2%glutaraldehydeinPBSwfor15min.
8. RinseinPBSw.
9. Wash3timeswithPBSwfor5min.each.
10. Washin1mlof50%PBSw:50%hybridizationsolution,followedby100%hybridizationsolutionforabout3min.eachstanding.
11. Replace900µloffreshhybridizationmixineachglassvial.
12. Prehybridizesamplesfor3hrsat65°C.
13. Heat200ngoftheRNAprobein100µlofhybridizationmixto95°Cfor5min.
14. Addtheprobe/hybridizationmixtotheembryos.Thefinalprobeconcentrationshouldbeabout200ng/ml.
15. Hybridizeovernightat70°Cinawaterbath.

InSituhybridization

DAY2.

1. Removehybridizationsolutionandadd800µlofprehybridizationsolution.Washfor5minutesat70°C.
2. Add400µlof2XSSC,ph4.5(withoutremovingprehybridizationsolution.)C.Repeattheadditionofthe2XSSCwashtwicemore.
3. Removethemixandwashtwice,30mineachtime,in2XSSCpH7/0.1%CHAPS70°C.
4. Washtwice,10mineach,inMaleicAcidBuffer(MAB;100mMmaleicacid,150mMNaCl;pH7.5)atroomtemperature.Washtwice,30mineachtime,inMABat70°C.
5. Washtwice10mineachinPBSatroomtemperature.
6. Wash5mininPBSwatroomtemperature.
7. Incubatetheembryosin1mlantibodybufferforatleast2hoursat4°Cwithrocking.
8. BMblock-mouseantibodybuffer2.5mlneededforeachsample:
   • 10%Goatserum(heatinactivated30minat56°C)
   • 1%boehringerblockingreagentinPBSw
   • Heatthemixtureat65°Cuntiltotaldissolution,filterthrough4.5micronfilters(severalmaybeneeded),thencoolonice.
   • Duringtheblockingstep,preabsorbetheantibodies.ThedilutionfortheAlkalinephosphataseconjugate(AP)is1/10000fromastockof150units/200µl(Boehringer).Dilutetheantibodyin1.5mlofantibodybufferandincubaterockingforatleast2hoursat4°C.Usethissolutiontoreplacetheblockingsolution.
9. Replacebufferwithdilutedantibodyandincubateovernightat4°Crocking.

InSituhybridization

DAY3.

1. Fastwashembryoswith0.1%BSAinPBSw.
2. Doanother5washeswith5mls0.1%BSAinPBSw(filltothetoptominimizeairbubble)rotatingfor45min.each.
3. Washtwice,30min.eachinPBSw.
4. TakeoutstainingsolutionstowarminRT.
5. WashtheembryosinAP1buffer(100mMTris9.5;100mMNaCl;50mMMgCl2)rockingfor10min,twotimeseach,atRT.
6. Replacewith1mlBMpurpleandrockslowlyinthedark.BMpurplestainingtakesafewhourstoseveraldays,ifnecessaryleaveat4°Covernightoruntilbackgroundappears.
7. StopstainingreactionbywashinginatleastthreechangesofPBS.
8. Afterstaining,dehydratethroughmethanolseries(25%,50%,75%,2x100%)andstoreinmethanolat-20°C.
9. Takepicturesafterplacingbackinmethanol.BMpurplebecomesmoreblueandintenseinmethanol.