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.

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