Wholemount in situ hybridization for the detection of RNA in C. elegans embryos

Whole-mountinsituhybridizationforthedetectionofRNAinC.elegansembryosGeraldineSeydouxandAndrewFire

AdaptedfromSeydoux,G.andFire,A.(1995).Whole-mountinsituhybridizationforthedetectionofRNAinC.elegansembryos.InC.elegans:ModernBIOLOGicalAnalysisofanOrganism.MethodsinCellBiology(ed.H.EpsteinandD.Shakes)AcademicPress,SanDiego.I.MaterialsA.Reagents-Anti-digoxigeninFabfragment,rhodaminelabeled(BoehringerMannheimCat#1207750)-Anti-digoxigeninFabfragments,alkaline-phosphataselabeled.(BoehringerMannheimCat#1093274)-BSA(FractionV,SigmaCat#A-7906)-CommercialBleach(5.25%Sodiumhypochloritesolution,Clorox)-DAPI(SigmaCat#D-1388)-DNAfromsalmontestes(Sigma,Cat#D-1626)-Formaldehyde(37%,Fisher,Cat#F79-500)-Formamide(BoehringerMannheimCat#100731)-Glycerol(BoehringerMannheimCat#100649)-Glycine(SigmaCat#G-4392)-Glycogen(BoeringherMannheimCat#901-393)-Heparin(Sigma,Cat#H-3393)-Hepes(BoehringerMannheimCat#242608)-Levamisole(Sigma,Cat#L-9756)-NaN3(SigmaCat#S-2002)-NBT:4-Nitrobluetetrazoliumchloride.(BoehringerMannheimCat#1383213)-p-Phenylenediamine(SigmaCat#P-6001)-0.1%Poly-L-lysinesolution(SigmaCat#P-8920).-Polyoxyethylene-SorbitanMonolaurate(Tween20;SigmaCat#P-1379)-ProteinaseK(BoehringerMannheim,Cat#161519)-TaqDNApolymerase(PromegaCat#M1861)-Tris(BoehringerMannheimCat#604205,812854)-TritonX-100(SigmaCat#X-100)-X-phosphate:5-Bromo-4-chloro-3-indolyl-phosphate.(BoehringerMannheimCat#1383221)

B.StocksolutionsDEPCtreatmentofsolutionsisnotneeded.Solutionsarestoredat-20oCunlessotherwiseindicated.-10xdNTPmix:Digoxigenin-11-dUTPpre-mixedwithothernucleotides(1mmol/ldATP,1mmol/ldCTP,1mmol/ldGTP,0.65mmol/ldTTP,0.35mmol/lDIG-dUTP).(BoehringerCat#1277065.)-10xPBS:80gNaCl,2gKCl,6.1ganhydrousNa2HPO4,2gKH2PO4,H2Oto1liter.Autoclaveandstoreatroomtemperature.-10xTaqBuffer:500mMKCl,100mMTris-HCl(pH9.0at25oC),1%TritonX-100.-20xSSC:3MNaCl,0.3MNa3Citrate-2H20.Storeatroomtemperature.-Mountingmedia:70%glycerol,1mg/mlp-phenylenediamine(pH9).

C.WorkingsolutionsThesesolutionsarepreparedonthedayofuse.-Formaldehydefixativesolution:1XPBS,0.08MHepes(pH6.9),1.6mMMgSO4,0.8mMEGTA,3.7%formaldehyde.-HybridizationbufferforCDNA-derivedprobes:100µg/mlautoclavedsalmonspermDNA,50µg/mlheparin,0.1%Tween20,50%formamide,5XSSC.[IfyouareusingWheatonjarsthatholdupto20slidesin150ml,youwillneedatotalof1literofHYBbufferforthepre-HYBandpostHYBwashes:Prepare1literofHYBbufferomittingtheDNA.AliquotHYBinto4bottlesasfollows:-400mlinonebottlelabeledHYBw/DNA.Add4mlof10mg/mlssDNAtothataliquot.-360mlinonebottlelabeledHYBwash-180mlinonebottlelabeled3:2.Add120mlofPTwtothataliquot.-60mlinonebottlelabeled1:4.Add240mlofPtwtothataliquot.]-Hybridizationbufferforoligonucleotideprobes:100µg/mlautoclavedsalmonspermDNA,50µg/mlheparin,0.1%Tween20,andappropriateconcentrationsofformamideandSSCasdescribedinsectionIIID.-Hypochloritesolution:1NNaOH,1:10dilutionofcommercialbleach.-PBT:1xPBS,0.1%BSA,0.1%TritonX-100-PTw:1xPBS,0.1%Tween20-Stainingsolution:100mMNaCl,5mMMgCl2,100mMTris,pH9.5;0.1%Tween20;1mMLevamisole.Levamisoleisapotentialinhibitorofendogenousphosphatases.-TTBS:150mMNaCl,50mMTris-HClpH7.8,0.1%BSA,0.1%Tween-20

E.Slidesandothermaterials-Carter"srubbercement(DennisonStationaryProducts).-Coverslipsforfreeze-cracking(No.11/2;24x50mm;ThomasScientificCat#6663K94)-Incubationdishes.Unlessotherwisenoted,allwashesandincubationsaredoneinWheatonstainingdishes(ThomasCat#8541-H15),whichcanhold20slidesin150ml.-Parafilmsquarescutto20x20mm.-Slides(75x25mm)[Cel-LineAssociatesInc.(tel:1-800-662-0973),Cat#10-2066,brownautoclavablecoating].Theseslideshave3squarewells(14x14mm)surroundedbyathinhydrophobiccoatingsimilarinthicknesstoaC.elegansembryo.Thiscoatingsupportsthecoverslipduringfreeze-crackingandfacilitatesincubationwithsmallvolumesofstainingsolutions.Onlythetwooutsidewellsareused.Wellsaresubbedwithpoly-lysineonthedayofuse:50µlofpoly-lysinesolutionisallowedtosettleonslidesfor10-20min;excesssolutioniswipedoffandslidesarebakedat60oCfor10min.

II.PROCEDUREA.Overview.AmixedpopulationofC.elegansembryosisattachedtomicroscopeslides,permeABIlizedbyfreezingandfixedwithmethanolandformaldehyde.Embryosarethenincubatedovernightwithadigoxigenin-labelledsingle-strandedDNAprobe,followedbyextensivewashestoremoveexcessprobe.Fluorescentorenzyme-linkedanti-digoxigeninantibodiesareusedtovisualizethehybridizedprobe.Theentireprocedurerequiresapproximately1.5daysfromharvestofembryostoprobevisualization.Thisprotocolisdesignedforembryos,butcanalsobeusedforlarvae/adults.BestresultshavebeenobtainedwhenlookingatRNAsexpressedintheadultgermline,butsomesomaticRNAshavealsogivennicepatterns.Modificationsnecessarytoworkwithlarvae/adultsaredescribedinsectionD.

B.Probesynthesis

Wehaveusedtwotypesofsingle-strandedDNAprobes:probesderivedfromclonedcDNAs,andsyntheticoligonucleotides.ThecDNA-derivedprobesareusedtodetectmRNAsderivedfromspecificgenes.TheseprobesaresynthesisedbymultiplecyclesofprimerextensioninthepresenceofdigoxigenindUTP,usingaclonedcDNAasatemplate(eg."asymmetricPCR",PatelandGoodman,1992).TheoligonucleotideprobesaresuitablefordetectingabundantRNAscontainingadefinedsequencesuchaspoly-A+RNAs(usinganoligo-dTprobe)andSL1-bearingRNAs(usingananti-SL1probe).Theseprobesareend-labeledwithterminaltransferaseanddigoxigenin-ddUTP.

1.Protocolforpreparationofsingle-strandedprobesfromclonedcDNA(fromPatelandGoodman,1992):a)2-5µgofplasmidDNAcontainingthecDNAinsertislinearizedusinganappropriaterestrictionenzyme.Forantisenseprobes,auniquerestrictionsite5"totheinsertisused.ThisdigestedDNAwillbeamplifiedusinganantisenseprimeratthe3"endoftheinsert.Forsense(control)probes,auniquerestrictionsite3"totheinsertisused.ThisdigestedDNAwillbeamplifiedusingasenseprimeratthe5"endoftheinsert.(Insertsofupto2kbarelabeledefficiently).b)DigestedDNAisextractedoncewithphenol/chloroform,oncewithchloroform,precipitatedwith3volumesof100%EtOH,andresUSPendedinTEatafinalconcentrationof100-200µg/ml.c)Thefollowingreagentsaremixedin0.5mlEppendorftube.water7.0µl10xTaqBuffer2.5µl25mMMgCl21.5µl10xdNTPmix5.0µlPrimer*(30ng/µl)5.0µlDigestedDNA(100-200µg/ml)2.0µlmineraloil40µl*ForcDNAsclonedinBluscript,weusethefollowingprimers(21mers):"T3"=5"-ACTAAAGGGAACAAAAGCTGG-3""T7"=5"-ACTCACTATAGGGCGAATTGG-3"d.Mixedreagentsareboiledfor5min,beforeadding2.0µlofa1:8dilutioninwaterof5units/µlTaqpolymerasestock(1.25unitstotal).e.Thelabellingreactionisincubatedfor35thermalcyclesasfollows:95°Cfor45seconds55°Cfor30seconds(lowertemperatureforprimerslessthan20nt)72°Cfor1minutef.75µlofH20isaddedtothereactionbelowtheoiland90-95µlofthedilutedreactionistransferredtoanewtube.g.10µlof1MNaCl,10µgofglycogen,and3volsof100%EtOHareaddedtothedilutedreaction.After30minat-70°C,thereactioniscentrifugedat15,000rpmfor10min.Thepelletiswashedin70%ethanol,dried,andresuspendedin300µlofhybridizationbuffer.h.Theprobeisboiledfor1-2hours.Thisstepreducesthelengthoftheprobeforefficientpenetrationofembryos.e.Probeproductionisassayedusingthefollowingprotocol.1µlofprobeinhybridizationbufferismixedwith5µlof5xSSC,boiledfor5min,andcooledonice.1µlofthismixtureisspottedonanitrocellulosestrip.Severaldilutionsofapre-labelledcontrolDNA(1ngto1pg/µl;BoeringherMannheim)arealsospottedforcomparison.Thestripisbakedfor30mininavacuumovenat80oC,washedoncein2xSSC,twiceinPBT,andblockedfor30minutesinPBT.Thestripisthenincubatedfor30-60minwithAP-antiDIGantibodydiluted1:2000inPBT.Afterthree10minwashesinPBT,andtwo5minwashesinstainingsolution,thestripisdevelopedinstainingsolutioncontaining4.5µlNBT/ml,3.5µlX-phosphate/ml.SpotsshouldbevisIBLewithinminutes.Spotintensitiesoftheprobeandcontroldilutionsarecomparedtodeterminetheconcentrationoftheprobe.f.Probescanbestoredat-20oCinhybridizationbufferforseveralweeks.

2.Synthesisofoligonuceotideprobes:Syntheticoligonucleotidesareend-labelledusingterminaltransferaseanddigoxigenin-ddUTP(BoeringherMannheimsellsan3"end-labelingkit[Cat#1362372];wehaveusedthesereagentsongel-purifiedoligonucleotides).Labeledoligonucleotideprobesareresuspendedto0.5µg/mlinhybridizationbuffer.ThepercentageofformamideandconcentrationofSSCinthehybridizationbufferareadjustedforeacholigonucleotidetogiveameltingtemperature(Tm)of52oC,usingthefollowingformula(Davisetal.,1986):Tm=16.6log[M]+0.41[Pgc]+81.5-B/L-0.65[Pf]whereMisthemolarconcentrationofNa(maximumof0.5),PgcisthepercentofGandCbasesintheoligonucleotide,Bis675forsyntheticoligosupto100basesinlength,Listhelengthoftheprobeinbases,andPfisthepercentconcentrationofformamide.ATmof52oCallowsforefficienthybridizationat37oC.

C.CollectionofEmbryos1.WormsaregrownonalawnofE.colistrainOP50onNGMagarplates(100x15mm).ForthewildtypestrainN2,eachplateisstartedwith20adulthermaphroditeswhichareallowedtogrowuntilalltheirProgenyhavestartedtolayeggs.Tensuchplatesyieldenoughembryosforapproximatelythirtyindividualwells.Chunksofagaronplatesshouldbeavoided,sincethesecaninterferewiththefreeze-crackingstep.2.Gravidhermaphroditesandtheirlaideggsarewashedofftheplatesinwater,andcollectedintoa15mlconicaltube.Aftercentrifugation(1700rpmforasufficienttimetopelletembryosandadults,approximately1min),mostofthewaterabovethepelletisremoved.3.Thewormpelletisresuspendedin10mlofhypochloritesolution,andincubatedatroomtemperaturefor3minuteswithsomeagitation.Wormsarerecoveredbycentrifugationandincubatedinfreshhypochloritesolutionforanother3minutes(Incubationtimesmayvarydependingonthetypeofbleachused).Embryosareprotectedbytheireggshellfromhypochloritedigestion,whilelarvaeandadultsaredissolvedbythistreatment.4.Whencarcassesoflarvaeandadultsarenolongervisibleinthedissectingmicroscope,embryosarewashedtwicein15mlofPBS,beforeresuspendinginasmallvolumeofPBS(0.5mlorless).

D.Permeabilizationandfixationofembryos1.Embryosaretransferredtothepolylysine-coatedslidesusingamicro-pipet.(15µlofembryosinPBSissufficienttocovera14x14mmsquarewell).Makesuretheembryosarenotformingclumps.Clumpscanbeseparatedbyblowingairontotheembryosthroughamicropipet.2.Embryosareoverlayedwithaglasscoverslipandtheslideisplacedinahumiditychamber.Repeatforallslides

Modificationofprotocolforlarvaeandadults:WashwormsinPBS,spinthemdown,resuspendinsmallvolumeofPBS,andspread5ulcontaining10to50wormsontothepolylysined14x14mmwell.CoverthewormswithacoverslipmakingsurethatthePBSspreadsontothepaintedpartoftheslidethuspullingthecoverslipdown.Youcanthenpressonthecoverslipeversolightlytoburstopenthehermaphrodites,soastoobtainbestpermeabilizationofthegermlineandembryos.Proceedwithfreeze-cracking(step3).

3.Slidesarefrozenbyplacingonanaluminumblockthathasbeenpre-cooledondryice.Thecoverslipsarethenquicklysnappedoff,andtheslidesareimmediatelyimmersedin100%methanolat-20oCfor5min.3.Slidesaretransferredto100%methanolatroomtemperaturefor5minandthenrehydratedatroomtemperatureasfollows:-One1minwashin90%MeOHinH2O.-One1minwashin70%MeOHinPBS.-One1minwashin50%MeOHinPBS.-Two5minwashesinPTwIFYOUDONOTPLANTODOTHEPROTEINASEKDIGESTIONSTEP,YOUCANSKIPTHESELASTTWOPTwWASHESANDGODIRECTLYFROM50%MeOHTOTHEFIXATIVESOLUTION(step5).4.Atthispoint,aProteinaseKdigestionstepcanbeincorporated.Suchastepmaybenecessarytoincreasetheaccessibilityoflowabundancemessagesexpressedafterthelima-beanstage(PeteOkkema,pers.communication).However,forearlierstages,proteinaseKtreatmentisnotrecommended.EachbatchofproteinaseKneedstobetitratedtodetermineproperincubationconditions.(Typically,a15minincubationina1µg/mlsolutioninPTwatroomtemperatureissufficientifneeded).ProteinaseKdigestionisstoppedbyincubatingfor2minin2mg/mlglycineinPTw,followedbytwo5minwashesinPTw.(Ifproteasedigestionisnotnecessary,proceeddirectlyfromstep3tostep5).5.Embryosarefixedbyincubationatroomtemperaturefor20mininformaldehydefixativesolution.6.Toremoveformaldehyde,embryosarewashedextensivelyatroomtemperatureasfollows:-Two5minwashesinPTw.-One5minwashin2mg/mlglycineinPTw.-Three5minwashesinPTw.E.Prehybridization1.Fixedembryosareincubatedfor10minina1:1mixofhybridizationbufferandPTw,followedbya10minincubationinundilutedhybridizationbuffer.(Theseincubationsaredoneatroomtemperature).Duringthistime,aseparate150mlaliquotofhybridizationbufferisheatedinaboilingwaterbathfor10min,andcooledonice.2.Embryosarepre-treatedinthisfreshly-heatedhybridizationbufferfor1-2hoursatthetemperaturetobeusedforprobehybridization(48oCforcDNA-derivedprobesand37oCforoligonucleotideprobes).

F.Hybridization1.cDNA-derivedprobescanbeusedundiluted(original300µl,appr.5µg/ml)oruptonine-folddiluteddependingontheabundanceofthetranscript.Ingeneral,theprobeisdiluted3-foldformoderatelyabundantmessages(eg.skn-1mRNA)anduptonine-foldforveryabundantmessages(eg.unc-54mRNA;lacZmRNAderivedfromamultiple-copyarray).Oligonucleotideprobesareusedataconcentrationof0.5µg/ml.2.Theprobeisboiledfor10minthencooledonice.3.Slidesareremovedfromthepre-hybridizationbufferandexcessbufferiswipedoffwhilekeepingtheembryoswet.4.20µlofdilutedprobeisaddedtoeachwell.Eachwellisthencoveredwithasquareparafilmcoverslip(20x20mm)whichissealedontotheslidewithrubbercement.5.Slidesareincubatedinasealedhumiditychamberovernightat48oC(forcDNA-derivedprobes)or37oC(foroligonucleotideprobes).6.Youcanalsoincubatethepost-HYBwashsolutions(HYBwash,3:2,1:4andPTw)inthesameincubatorovernight.

E.Post-hybridizationwashesAfterhybridization,parafilmcoverslipsareremovedwithforcepswhilekeepingtheslidessubmergedinhybridizationsolution.Embryosarethenwashedwithgentleagitation(eg.40-60rpminshakerwith17cmrADIus)asfollows:1.WashesforcDNA-derivedprobes:(HYBsolutionusedinwashesdoesnotrequiressDNA)-Two15minwashesinhybridizationsolutionat48oC.-Two15minwashesin3partshybridizationsolution/2partsPTwat48oC.-Two15minwashesin1parthybridizationsolution/4partsPTwat48oC.-Two15minwashesinPTwat48oC.-Two20minwashesinPBTatroomtemperature.2.Washesforoligonucleotideprobes:-One10minwashinhybridizationsolutionat37oC.-Two10minwashesinTTBSatroomtemperature.

F.ProbeDetectionTwotypesoflabelsareavailablefordetection:Markerenzymessuchasalkaline-phosphatase(AP)andfluorescenttagssuchasrhodamineorfluorescein.AP-mediateddetectionishighlysensitivebutresultsinasignalwithlimitedsub-cellularresolution.Incontrast,fluorescentdetectionisgenerallylesssensitivebutallowsmoredefinedsub-cellularresolution.Forthesereasons,weprefertouseAP-mediateddetectiontoidentifycellsexpressingspecificmRNAs,andfluorescent-detectiontodeterminethesub-cellularlocalizationofrelativelyabundantRNAs.

1.AP-mediateddetection:a.30µlofthedilutedAP-anti-DIGantibodyconjugate(1:2500inPBT,0.3units/ml)isappliedtoeachwell.Eachwellisthencoveredwithasquareparafilmcoverslipandincubatedinahumiditychamberfor2hoursatroomtemperature.b.Embryosarewashed4timesfor10mininPBT.(Ifanoligonucleotideprobewasused,embryosarewashedtwicefor10mininTTBSinstead.)c.Embryosareincubatedtwicefor5mininfreshly-madestainingsolution.d.30µlofstainingsolutionwith4.5µlNBT/ml,3.5µlX-phosphate/mland1µgDAPI/mlisappliedtoeachwell.Slidesarekeptinthedarkduringthecolorreaction.Thesignalshouldappearafter20mintoonehourdependingontheprobe.Thecolorreactionismonitoredunderthemicroscopetoavoidbackground,andisstoppedbywashingembryostwiceinPBS.e.Embryosaremountedin5-10µlofa70%glycerolsolutionandcoveredwithaglasscoverslip.

2.Fluorescencedetection.a.30µlofRhodamine-anti-DIGantibodyconjugate(0.2mg/mlwith1µgDAPI/ml)isappliedtoeachwell.Eachwellisthencoveredwithasquareparafilmcoverslipandincubatedinahumiditychamberfor2hoursatroomtemperature.b.Embryosarethenwashed4timesfor10mininPBT.(Ifanoligonucleotideprobewasused,embryosarewashedtwicefor10mininTTBSinstead.)Forincreasedsensitivity,asecondaryantibodycanalsobeused.e.Embryosaremountedin5-10µlofmountingmediaandcoveredwithaglasscoverslip.

III.DOUBLE-LABELLING

Tofacilitatetheidentificationofcellsinwholemountembryos,itisoftenusefultolabelspecificcellsusingantibodiesorothermarkers.Here,wedescribetheuseofananti-PgranuleantibodytolabelthePcells(P1-P4)andtheirgermcelldescendants(Z2andZ3)(StromeandWood,1982)inembryosthathavebeensubjectedtotheinsituhybridizationprotocoldescribedabove.Intheory,anyantibodycouldbeusedinasimilarmanner,providedthatitsepitopeisnotdestroyedbythehybridizationprocedure.1.Aftercompletionofthedetectionstepbutbeforemountingtheembryosinglycerol,theembryosaresubjectedtothree10minwashesinTTBS.2.Embryosareincubatedfor2hoursatroomtemperaturewithanFSE-labeledanti-Pgranuleantibody(diluted1:400inTTBS;weuseFSE-conjugatedOIC1D4fromJanetPaulsenandSusanStrome).3.EmbryosarewashedtwiceforfiveminutesinTTBS,beforemountingin5µlofmountingmedia.Pgranulestainingshouldbeseeninmostembryos.Strongalkalinephosphatasestainingcanquenchthefluorescenceoftheanti-Pgranuleantibodyconjugate.

IV.INTERPRETATIONA.GeneralityofthetechniqueWehaveusedthisprotocoltoanalysetheexpressionpatternof21genesexpressedduringembryogenesis(SeydouxandFire,1994).WefindthattheprotocolallowsthevisualizationofRNAinwell-preservedembryosfromtheone-cellstagetothepretzelstage(seeFig.1).Ingeneral,RNAsaredetectedinthecytoplasmofcells,withtheexceptionofembryonicallytranscribedRNAswhichcansometimesbedetectedinnucleiwhentheyarefirsttranscribedinearlyblastomeres.LowabundanceRNAsexpressedafterthelimabean-stagecanbedifficulttodetect;proteinaseKdigestionofembryospriortohybridizationcanbehelpfulinsuchcases(PeteOkkema,pers.comm.).AlthoughtheprotocolwasdevelopedtodetectRNAsinembryos,preliminaryresultssuggestthatitisalsobeapplicablefordetectingRNAsinlarvaeandadults(G.S.andA.F.,unpublisheddata).

B.lacZfusionRNAsBecauseoftheirgreatabundance,RNAsderivedfromchromosomallyintegratedlacZfusionsareanexcellenttargetforinsituhybridization.Whenfirsttranscribed,theseRNAsaccumulateintwonuclearfoci,whichmaycorrespondtothesitesoftranscriptiononthetwohomologouschromosomesthatcarrythearray(SeydouxandFire,1994).Theappearanceofthese"doubledots"canhelpdeterminetheearliestonsetoftranscriptionforageneofinterest.DoubledotscanoccasionallyalsobeseenforendogenousRNAs,butareingeneralmoredifficulttodetect.IncontrasttoendogenousRNAsthatquicklyaccumulateinthecytoplasmaftertheirinitialappearanceinthenucleus,lacZfusionRNAsremainpredominantlynuclearandappearquitelabileuntilthe26-cellstage(SeydouxandFire,1994).Afterthatstage,lacZfusionRNAsaccumulateinthecytoplasmandbecomemorestable,oftenperduringlongerthanendogenousRNAs.ThisbehavioroflacZfusionRNAsmaybeduetothelong,intron-lesscodingregionofthelacZgene.

C.Backgroundvsauthenticstaining.Acommonproblemassociatedwiththeprotocolpresentedhereisthehighincidenceofnonspecificstickingoftheprobetoembryos.Often,upto50%ofallwellsinanexperimentexhibitsomeformofnon-specificstaining.Thisproblemmaybeduetovariabilityinthepermeabilizationofembryosintroducedduringthefreeze-crackingstep.Fortunately,thisnon-specificstainingiseasilydistinguishedfromauthenticstaining.Non-specificstainingusuallyappearswithin10minutesinthecolorreactionasdarkpurplepatchesonthesurfaceofembryosorinnuclei.Thebestwaytodistinguishauthenticstainingfromnon-specificstainingistocomparestainingpatternsobtainedfrombothantisenseandsenseprobes.Anystainingcommontobothprobesislikelytobeduetonon-specificbackground.Inourexperience,successfulhybridizationwithsenseprobesyieldsembryoswithnostainingatall.

VI.Trouble-shooting

Inthetablebelow,welistsuggestionstolimittheoccurrenceofnon-specificstainingandothercommonproblems.

Problem	Cause	Solution
Non-specificstaining:patchyoruniformstainingonthesurfaceofembryos,and/orstainingofallnuclei.	Embryoclumping	Limithypochloritetreatmenttominimumamountoftimeneededforremovalofadultandlarvalcarcasses.Beforetransferingtheembryosuspensiontotheslide,useamicro-pipettoblowairinthesuspensiontobreakanyclumps.
	Poorfreeze-crackingstep	EmbryosshouldbeinsmallvolumeofPBS(10-20µlfora14x14mmwell).Avoidhavingsmallpiecesofagarontheslide.Usemorethanonewellforeachexperiment.(4wellsusuallyguaranteesatleastonegoodfreeze-crack.)
Non-specificstaining:faintstainingalloverembryos	Excessiveprobeconcentration	Reduceconcentrationofprobe(Concentrationsintherangeof0.5-2.5ng/µlarerecommendedformostRNAs.)
Non-specificstaining:faintstaininginextrudedportionsofembryos	ThisappearstobeanintrinsicpropertyoftheBoehringerantidigoxigeninAP-conjugatedantibody.	Onlyintactembryosshouldbeexamined.
Nosignal	Oversquashedembryos	Donotpresstoohardonthecoverslipwhenfreezingtheembryos.
	OverorunderProteinaseKdigestion	Varyconcentrationand/ordurationofproteinaseKdigestion.Note:ProteinaseKdigestioncanboostthesignalobtainedfromRNAsinlima-beanandolderembryos.ProteinaseKdigestionisnotrecommendedforpregastrulationembryos.
	Lowprobeconcentration	Increaseprobeconcentration.
Noembryosleftonslide	Poorfreeze-crackingstep	ReducevolumeofPBSusedinfreeze-crackingstep
	Notenoughpoly-lysine	Slidesarebestwhensubbedwithpoly-lysinesolutionthedayoftheinsitus.

Acknowledgments:

WearegratefultoNipamPatelwhoencouragedustodevelopinsituhybridizationforC.elegansembryosandgenerouslysharedhisprotocolsandexpertise.WethankTomEvans,DavidGreenstein,VincentGuacci,MikeKrause,ShoheiMitani,PeteOkkema,NeelaPatelandJorgeMancillasforadvice,SusanStromeforheranti-Pgranuleantibodies,DavidBird,DaveHsu,EdwardKipreos,VerenaPlunger,JimPriess,AnnSluder,DeborahRoussellandKarenBennett,PattyWohldmannandBobWaterstonforDNAclones,andBillKellyandPeterOkkemaforcriticalreadingoftheprotocol.

Mostofthisprotocolisderivedfrommethodsdescribedinthefollowingreferences:

Patel,N.H.andGoodman,C.S.(1992).Preparationofdigoxigenin-labeledsingle-strandedDNAprobes.InNon-radioactiveLabelingandDetectionofBiomolecules.(ed.C.Kessler).Springer-Verlag,Berlin.

Tautz,D.andPfeifle,C.(1989).AnonradioactiveinsituhybridizationmethodforthelocalizationofspecificRNAsinDrosophilaembryosrevealstranslationalcontrolofthesegmentationgenehunchback.Chromosoma98,81-85.