In Situ HYBRIDIZATION TO TISSUE SECTIONS

PARAFFINSECTIONSPreparationofembryos(¾10hr)forparaffinsections

• Washembryosoffcollectionplatesontoanylonscreenwith0.4%NaCl/0.3%TritonX-100.Rinsewelltoremoveallfoodandyeast.Blotwelltoremoveexcessliquid.
• Dechorionateembryosinfreshlydiluted50%chloroxfor3atroomtemperaturewithgentleagitation.RinsewithNaCl/Triton;blotwell.
• TransferdechorionatedembryostoeitherbufferAorB(dependingontheirage):
  • BufferA(0-2,1-3hrembryos)
  • 2.5ml8%paraformaldehyde(fresh)
  • 2.0ml1XPBS
  • 0.5mlDMSO
  -or-
  • BufferB(2-4,3-5hr,etc.)
  • 2.5ml8%paraformaldehyde
  • 0.5ml1XPBS
  • 2.0mlDMSO
• Add5mlheptaneandshakevigorouslyfor20(BufferA)or30(BufferB).
• Transferto10ml90%MeOH/EGTA(9mlMeOH+1ml50mMEGTA,pH8)+10mlheptane
  • TheMeOH/EGTA/heptanemixshouldbeprechilledto-70degreesCondryice.Ittakesawhiletocomedowntotemp.soplaceitondryiceabout1hrearlier.
  • Onewaytotransfertheembryostothecoldmixistoaspirateofftheheptane(top)andparaformaldehyde(bottom)phases(theembryoswillbeattheinterface),pourtheembryosoutontoascreenandharvesttheembryoswithapaintbrush.
  • Alternatively,removebothphasescompletelyfromtheoriginaltubeandthenpourthecoldmixontotheembryos.
  Shakevigorouslyondryicefor:10(0-2,1-3,3-5,4-6hrembryos)15(5-7,6-8and8-10hrembryos)
• Rapidlywarmtheflaskbyswirlingunderhotrunningtapwater.Thedevittellinizedembryoswillsinktothebottom.Transfertheembryostoavialwithawideborepipettip.Wash3Xwith90%MeOH/EGTA.
• Rehydrateembryos(0-2and1-3hrembryosneednotberehydratedthroughparaformaldehyde,justleavethemintheappropriateMeOH/PBSsolutions),10ineachsolution:MeOH/EGTA:1XPBS:4%Paraformaldehyde9:1:09:0:17:3:07:0:35:5:05:0:53:7:03:0:7
• Fixanadditional15in4%paraformaldehydewithoutagitation.Atthispoint,embryosmaybestainedwithDAPIorFluorochromes.Forsectioning,rinsethoroughlywithPBS.
• Dehydratethrough30%,50%and70%EtOH;10ineachsolution(embryosmaybestoredatthispointat-20degreesCforatleastaweek).

• Completethedehydration:90%EtOH,1095%EtOH,10100%EtOH,10100%EtOH,10Xylenes,10Xylenes,10Transfertheembryostoasieveandpatdry.Thesievemaybeputinabeakerwithxylene(becauseoncetheembyosareinxylenetheyarehardtosee).
• TransfertoXylene/paraffin(1:1)@58degreesCandallowthemtositinthismixturefor20(embryos¾6hr)or30(embryos>6hr).Becarefultonotletthetemp.dropbelow56degreesCorthewaxwillhardenrapidly.Donotletthetemp.goabove62degreesCorthewaxwillnotpolymerizecorrectly!
• Placeembryosinthemold(ScientificProd.7275-1;peel-awaydisposable,8x8mm):
  • CutofftheendofaPasteurpipetteandheatitbrieflyinaflame.Plungeintomeltedwaxtolowerthetemperatureofthepipette.
  • Drawupasmanyembryosaspossibleinassmallavolumeaspossibleusingthewarmedpipette.
  • Expeltheembryosquicklyandcarefullyontothebaseofapreheatedplasticembeddingmold.Keepthedropinthecenterofthemold.Ifittouchestheedges(anditwill!),swirlthemoldsothattheembryoswillsettleinitscenter.
  • Allowtheembryostosettleforabout10.Whentheyarealmostlongitudinal,removethemoldfrom58degreesC.
  • Watchthewaxset.Whenaskindevelopsonthetop,quicklypourmeltedwaxdownthesideofthemolduntilitisatleasthalffull.Letitharden.Ifyoupourthewaxintoosoon,theembryoswillbeunsettled(andthereforenolongerinthecen-ternorlongitudinallyoriented).Ifyoupourthewaxintoolate,thelayercontainingtheembryoswillnotsticktotherestoftheblock,makingsectioningimpossible.
• Blocksmaybestoredat4degreesCforatleast6monthsandprobablylonger.

• Trimtheblocktoatrapezoidalshapewitharazorblade(trytocutoffaslittleaspossi-ble).Thetrapezoidalshapefacilitateslaterseparationofsections,onefromanother.
• Mounttheblockontoawoodenspecimenholderusingmeltedwax(justmelttheshavingsfromthetrimmedblockwithahotspatulaandquicklypresstheblockintothepoolofmeltedwax).Letithardenabout5.
• Mountthespecimenholderontothemicrotomewiththesmalleredgeontop.
• Cutsections.Itispossibletomakesections4micronsthick,buttheyteareasily.Sixmicronsectionsareeasilymanaged.Theknifeshouldbethoroughlycleanedwithxyleneinbetweenblocks.DoingthiswillpreventmanyproblemsTroubleshooting
  • Possibleproblem:Sectionstearastheyaresliced.Potentialremedies:Theremaybeapieceofparaffindustontheknife.Removeitwithabrush.Ortheremaybeanickontheblade.Changethepositionoftheknife.
  • Possibleproblem:Sectionssticktothetopoftheblockasthemicrotomereturnstoitsstartingposition.Potentialremedies:Theblademaybedirty.Carefully(withupwardstrokes)cleanitwithxylene.Ortheknifemaybeatabadangletotheblock.Adjustituntiltheproblemissolvedoruntilitisobvioustheknifeangleisnttheproblem.Ortheknifemaybetoowarm.Suspendachunkofdryiceovertheblock(ringstand).
  • Possibleproblem:Sectionsaredeformedastheyresliced.Potentialremedies:Slicingtoofast,slowdownthestroke.Orknifeand/orblockaretoowarm,coolwithdryice.
  • Possibleproblem:Sectionscurlupastheyaresliced.Potentialremedy:Checktheangleoftheknife.Orknifeand/orblockaretoocold.

• PutasmalldropofddH2Oonapolylysine-subbedslide.Thedropshouldbelargeenoughthatwaterisvisiblearoundtheperipheryofthesection.
• Placethesectionontopofthedrop.
• Lettheslidesdryona45degreesCslidewarmerfor6hrtoovernight.Theslidewarmershouldbeinadust-freearea.Asslidesdry,wrinklesinsectionsshouldflattenout.

• Dewaxsectionsinxylene,2X10.
• Rehydratethrough100%,95%,80%,60%and30%EtOH;1ineachsolution.
• Incubatein0.2NHCl,20.
• RinseinddH2O,30".
• Incubate@70degreesCin2XSSC(preheatedto70degreesC),30.
• RinseinddH2O.
• Pronasetreat:-Removetheslidesfromtherackandplacehorizontallyinatray.-WithaPasteurpipet,carefullydrip‰0.25mg/mlpronasesolutionontothesectionsandincubate10(thheincubationtimestartswithtreatmentofthefirstslide).-Drainoffpronaseandreplaceslidesintherack.
• Stopthepronasereactionbyinhibitionwith2mg/mlglycineinPBS,30"then1XPBS,2X30"
• Fixin4%paraformaldehyde,20.
• Rinsein1XPBS,2X4.
• Acetylation(dounderthehood):-Placerackedslidesin500ml0.1MTriethanolamine,pH8.0(fresh;titratewithHCl).-Whilestirringrapidly,add1.25mlaceticanhydridedropbydroptoensurepropermixing.Incubate10duringwhichtimethestirringmaybesloweddown.
• Rinsein1XPBS,2X3.
• Dehydratethrough30%,60%,80%,95%and100%,EtOH;2ineachsolution.
• Airdry.

• Collectanddechorionateembryosasdescribedforparaffinsections.
• EmbedunfixedembryosorlarvaeimmediatelyinO.C.T.
• PlaceadropofO.C.T.directlyonthechuck.
• ImmersethechuckinliquidnitrogenbutdonotletthenitrogencoverthetoportouchtheO.C.T.
• RemovethechuckwhentheO.C.T.startstoturnwhitebutbeforeitscompletelyfrozen.
• Placeasecond,smaller,dropofO.C.T.ontopoftheunfrozenportionofthefirst.
• Quicklyplacetheembryosinthecenterofthedrop.Withapaintbrush,gentlymovetheembryosbackandforthuntiltheyareevenlydistributedoverthemiddleportionofthedrop.
• Placethechuckbackintoliquidnitrogenuntiltheseconddropiscompletelyfrozenasbefore(again,donotletthenitrogentouchtheO.C.T.).
• Putthechuckwithembryosinthecryostatsotheycancomeuptotemperature.

• Takethechuck(plusembryos!)outofthecryostat(theyshouldbeatcryostattempera-ture,-14to-19degreesC).
• Quicklytrimtheblockintoarectanglewitharazorblade.
• Putthetrimmedblockbackintothecryostat.Iftheblockfallsoffthechuck,glueitbackonwithsomefreshO.C.T.
• Placethechuckintothemicrotomehead.
• Slice.Sectionsaremountedontoslidesastheyaresliced.Pickupsection(s)directlyofftheknifeontoagelatinsubbedslide.Youwonthavetotouchthesections,theywill"jump"offtheknifeontotheslide.Pickingupsectionswilltakeabitofpractice.-6-8micronsectionsarefairlyeasilymanaged-put1-3sectionsoneachsubbedslide
• Airdrytheslidesforatleast20.
• Fixwith:4%paraformaldehyde203XPBS51XPBS51XPBS530%EtOH260%EtOH280%EtOH595%EtOH2100%EtOH2
• SectionsmaybestoredatRTforatleastamonth.

  TroubleshootingProblemsandtheirsolutionsareessentiallythesameasforparaffinsections.However,theknifeshouldnotbecleanedwithxylenes.Changesintemperatureshouldbeachievedbyregulatingthecryostattemperature.Inaddition,iftheguide-plateisinabadpositionyoumaygettornormangledsections,sectionsthatcurlupanddontcomedowntheknifeorsectionswhichsticktotheguideplate.

• Incubatein0.2NHCl,20.
• RinseinddH2O,30".
• Incubate@70degreesCin2XSSC(preheatedto70degreesC),30.
• RinseinddH2O.
• Pronasetreat:-Removetheslidesfromtherackandplacehorizontallyinatray.-WithaPasteurpipet,carefullydrip0.25mg/mlpronasesolutionontothesections.-Theincubationtimestartswithtreatmentofthefirstslide.-Drainoffpronaseandreplaceslidesinrack.
• Stopthepronasereactionbyinhibitionwith2mg/mlglycineinPBS,30",then1XPBS,2X30".
• Fixin4%paraformaldehyde,20.
• Rinsein1XPBS,2X4.
• Acetylate(doinfumehood):-Placerackedslidesin500ml0.1MTriethanolamine,pH8(fresh;titratewithHCl)-Whilestirringrapidly,add1.25mlaceticanhydridedropbydroptoensurepropermixing.Incubate10duringwhichtimethestirringmaybesloweddown.-Ifgelatinslidesareused,acetylationisunecessaryandstep19maybeomitted.
• Rinsein1XPBS,2X3.
• Dehydratethrough30%,60%,80%,95%and100%EtOH;2ineachsolution.
• Airdry.

• Lyophilize25ml(=25mCi)eachof3H-dATP,dCTP,dGTPandTTPinanEppendorftubethathasbeenrinsedwithEtOH.Minimizethedryingtime(¾1hinSpeedVac)bydividingintosmalleraliquots,ifnecessary.
• Tothetubeoflyophilized3H-dNTPs*,add:+2.5ml10XNTB+0.5mgDNA+H2Oto24mlResuspendbyvortexing.*Ifnotallnucleotidesaretritiated,addcoldnucleotidesto25mM
• Add0.5-2.0mldilutedDNaseI(1:400dilutionof1mg/mlstock).IfthepreciseamountofDNasetouseisnotknownforaspecificDNAfragmentorbatchoftheenzyme,setupseveralreactions(0.5,1.0,2.0ml)inparallel.
• Add10UPolI.
• Incubate45@roomtemperature.
• Stopthereactionbyadding25ml50mMEDTA(pH8),onice.
• Determinethe%incorporationbyTCAprecipitation:-Add1mlreactionmixtureto100ml0.5mg/mlcarrierDNAina7mlglasstube-Spot10mlonaGF/Cfilterdisk-Totheremaining90ml,add5mlcold10%TCA;incubate10onice-FilterontoGF/C,washwith10%TCA,washwith95%EtOHanddry-20-50%incorporationisgoodandcorrespondstoaS.A.‰1-2x108dpm/ug-probewithincorporation¾10%shouldnotbeused
• UnincorporatednucleotidesmayberemovedonaStratagenePushColumn.
• Probesizemaybecheckedona8%acrylamide/7MUreasequencinggel.-Prerun200V,30-Load:105cpm/sampleto10mlformamide,boil1,chillandadd2.5ml5Xloadingbuffer(0.25%BPB,0.35%XC,15%Ficollin5XTBE)-End-labeledDNAfragments(e.g.,pBR322/HinfI)maybeusedassizeMarkers-Run200V,‰4h(BPBabout2/3downthegel)-Soak20indistilledwater-Impregnate15-30inFluorohance(RPI;orcomprableenhancer)-Dryandautoradiograph-Optimalsize75-150bp
• Preparationofprobeforhybridization(useat2-20ng/ml)-Precipitate125mgcarrierDNAper0.5mg3H-probe(dryice,spin10,70%EtOHwash,airdryinvacuo2)-Resuspendin10mlddH2O-Denatureprobebyboilingfor2,chillandquickspin-Add300mlhybridizationbufferandstoreonicebeforeuse.Probe(inhyb)maybestoredseveralmonths@-20degreesC.Beforeusing,boil2andchillonice.

• PlasmidtemplatesshouldbeRNase-freeCsCl-bandedDNA.LinearizeDNAwithanenzymethatcleavesdownstreamoftheinsert.Digestwith200mg/mlProteinaseK(30@37degreesC),phenol-Sevagextract(2X)andNaOAc/EtOHppt.ResuspendinTE@1mg/ml.Checkongelforcompletenessofdigestion.
• Transcriptionreaction(25ml):9.5mlDEP-H2O5.0ml5XTB1.0mllinearizedDNAtemplate1.0ml10mMrATP1.0ml10mMrCTP1.0ml10mMrGTP1.0ml0.5mMrUTP1.0ml0.75MDTT1.0mlRNase-Block(25U/ml)2.5ml35S-rUTP(100mCi)1.0mlT3orT7polymerase(10U)Vortex,spindownandincubate1-2h@30degreesC.Incorporationshouldbe70-90%.
• Determine%incorporationbyTCAprecipitation:-Add1mlreactionmixtureto100ml0.5mg/mlcarrierDNA-Spot10mlonaGF/Cfilter-Totheremaining90ml,add5mlcold10%TCAandincubate10onice-FilterontoGF/Cpaper,washwith10%TCA,rinsewith95%EtOHanddry-Fractionincorporated=cpm(TCA)/cpm(spot)x10-Calculatethemassofprobesynthesized:ngprobe=fractionincorporatedxinput35S(inCi)/SpecificactivityofUTP(Ci/mmol)x340x106ng/mmolx4
• Add1mlRNase-freeDNase(1U/ml)tothe24mlreactionmixtureandincubate30@37degreesC.
• Stopthereaction:+60ml10mMEDTA,10mMTris-HCl(pH7.4),0.2%SDS+1.67ml5MNaCl+1.0ml1MDTTIncubate10-15onice
• Hydrolyzetheprobe(idealfragmentlength=75-150NTs)byadding+2ml10NNaOH.Incubate45onice.
• Neutralizethereaction:+20ml2MHEPES(unpHd).
• Phenol-Sevagextract.
• RemoveunincorporatednucleotidesoveraStratagenePushColumn.-Equilibratecolumnwith70mlSTE+20mMDTT-Loadsample(‰110ml),pushthrough-Washwith70mlSTE+DTT
• Count1ml.
• Checklengthofhydrolyzedandunhydrolyzedprobeona6%acrylamide/7MUreagel.End-labeledDNAfragmentsmaybeusedasmarkers.
• Probesarestoredas5Xconcentrates.PrecipitatewiththeappropriateamountoftRNAasdeterminedbelow.Theamountofprobeusedperslideis0.3-1.0ng/ml/kb(wherekbreferstothelengthoftheunhydrolyzedprobe).Resuspendtheprobein20mMDTT/50%Formamide(1/5thvolumeofthefinalhybsolution)andstore@-70degreesC.mgtRNA=(nmollabel)(fractionincorporated)(350ng/nmol)(4)(0.3ng/ml)(kb)

HYBRIDIZATIONANDWASHES

Hybridization

• Pretreattheslides.
• Mixprobewithappropriatebuffer.
• Placehybmixtureontotheslides:Layslideshorizontallyonablacksurface.Apply10-15mlprobesolutionalong-sideoneedgeofthetissue.Usingforceps,carefullyloweran18x18mmcoverslipfromoneside,evenlydistributingthehybsoastocovertheentiretissuesection.Avoidairbubbles!
• Toavoidevaporation,sealtheedgesofthecoverslipwithrubbercementusingabrokenoffPasteurpipetorsyringe.Allowthecementtodry.
• Incubateslidesinahumidchamber18-24hat37degreesC(DNAprobe)or50degreesC(Riboprobe).

• Foreveryrackofslides,prepare2LDNAWashBuffer(for5washes).
• Afterhybridization,gentlypeelofftherubbercementsealwithapairofforceps.Standtherackedslidesinthefirstwashfor5@roomtemperature(RT).Carefullypickupeachslide(thecoverslipshouldfallrightoff)anddipacoupleoftimesinthewash.
• Immediatlelytransfertheslidetoarackinthesecondwashandincubate10-15@RT.DoNOTlettheslidedryout!
• Transfertheslidesinthethirdwashintoa37degreesCwaterbathfor18-24h.Duringthistime,changethewashbuffertwoadditionaltimes.
• DehydratetheslidesthroughEtOH(@RT):70%(2x5)and95%(2x5).
• Airdry.

• Foreveryrackofslides,prepare2LeachofWDTTandNTEwashbuffers.
• RemovecoverslipsasdescribedaboveforDNAprobesbutuseWDTTwashbufferwhichhasbeenpreheatedto50degreesC.
• Wash4-6hinWDTT@50degreesC.
• RNasetreat:Rinseslides2x5inNTE@37degreesCIncubate30in20mg/mlRNaseAinNTE@37degreesCRinse2x5inNTE@37degreesCWash1hinNTE@37degreesC
• WashinWDTT14-18h@50degreesC.Changethewashbufferonceduringthistime.
• DehydratetheslidesthroughEtOH:70%(2x5)and95%(2x5).
• Airdry.

• Preparetheemulsionbyplacinganaliquotina45degreesCwaterbathuntilmelted(nolongerthan10-15).Slowlypourtheemulsionintoaprewarmeddipper.Addanequalvolumeof45Cwaterandgentlyinvertthedippertomixtheemulsion.
• Placeametalorglassplateontoatraycontainingicetopromotequicksolidificationoftheemulsionontheslide.
• Diptheslidesindividuallyintotheemulsion(@45degreesC).Wipethebackoftheslidewithtissuepaperanddrainexcessemulsionbyholdingtheslideverticallyonapapertowel.
• Laytheslideontheprecooledsurfaceandallowtheemulsiontosetfor‰10.
• Transfertheslidestoaverticalpositioninadryingrack.
• Allowtheslidestodryinalight-proofplacefor„2h.Puttheslidesintoalightproofboxandexpose,dessicated,@4degreesCfortheappropriateamountoftime.Iuseaslideboxcontainingadryingagent(Drierite)sealedwithtapeandwrappedinaluminumfoil.

• RemovetheslidesfromtherefrigeratorandallowthemtocometoRT.
• Setupthreestainingdishescontainingdeveloper,waterandfixer.Theemulsionisveryfragilewhenwet.Itisimportantthatallthesolutionsbeatthesametemperatureduringthedevelopingprocess.Precoolallthesolutionsto15degreesCinawaterbathoronice.PlacethesolutionsatRTfordevelopingandwashing(allowedtowarmslowlyatRT).
• Placetheslidesinarackandimmerseinto15degreesCKodakD-19Developerfor4.
• Transferracktoastopbath(15degreesCwateror2%aceticacid)for30.
• FixtheslidesinKodakFixer(15degreesC)for5.
• Thelightsmaynowbeturnedon.
• PlacetheslidesbackinthewaterandletstanduntiltheyreachRT(atleast20degreesC).
• Rinse1-2timesinddH2O.
• StaintheslidesinGiemsa1-3(DNAprobes)or3-5(Riboprobes).Giemsadisplaysalotofbatchvariation,sotestoneslidebeforestainingthemall.Rinsebydippingintowaterandwipestainoffthebacksoftheslides(EtoHhelps).
• Airdry.
• Mounttheslideswith1-2dropsofPolymountorImmersionoil.

Aceticanhydride(SIGMAA6404)

Bleach(anycommercialproduct)

BovineSerumAlbumin(SIGMAA7906,FractionV)

CarrierDNA(SIGMAD1626,TypeIIIfromSalmontestes):Preparea10mg/mlstockinTE.Sheartoanaveragesizeof50-500bpbyautoclavingin100mlaliquotsfor20.CarrierRNA(SIGMAR1753,E.ColiStrainW,TypeXX):Preparea10mg/mlstockinTE.Phenolextract(2X)andNaOAc/EtOHppt.tocleanup.

100XDenhardts:2%ofeachBovineSerumAlbumin(SIGMAA7906,FractionV),Ficoll400(SIGMAF4375)andPolyvinylpyrrolidene(SIGMAPVP360).Store@-20degreesC.

50%(w/v)DextranSulfate(Parmacia17-0340-02):Mix110gdextransulfatein130mlsterilewater(takesawhiletogointosolution).Store@4degreesC.

DMSO(SIGMAD5879)

DNAHybridizationBuffer50%deionizedformamide0.6MNaCl1XDenhardts10mMTris-HCl,pH7.51mMEDTA20%Dextransulfate2-20ng/mlprobe

DNAPolymeraseI(BRL,NEB,etc.)

DNAWashBuffer-For1L:0.6MNaCl=120ml5MNaCl20mMTris-HCl,pH7.5=20ml1MTris-HCl,pH7.51mMEDTA=2ml0.5MEDTA50%Formamide=500mlFormamide

DNaseI(Worthington/CalbiochemDPFF):Preparea1mg/mlstockin10mMTris-HCl,pH7.5/10mMMgCl2.Freezein20mlaliquotsandthawonlyonce.EachbatchofDNasewillprobablyneedtobetitratedalongwiththeamountrequiredforoptimalradiolabelingofeachspecificDNAfragment.

1MDTT(SIGMAD0362,FW154.2)

0.5MEDTA,pH8(FW372.2)

50mMEGTA,pH8(FW380.4)

Emulsion[KodakNTB-2/Cat1654433(3H)orKodakNTB/Cat1654425(35S)]:Theemulsionisverysensitivetolightandshouldonlybehandledinthedarkunderredsafelight(KodakSafeLightFilterNo.2/Cat1521525,15Vlightbulb)conditions.Theemulsionisgelatin-basedandisasolidatroomtemperature.Dispensein15mlaliquotssincerepeatedfreeaing/thawingleadstoelevatedbackgroundlevels.Istoretheemulsionin50mlpolypropylenetubeswhichIsubsequentlyuseasthedippingchamber.Wrapthetubeinaluminumfoilandstoreat4degreesC(doNOTfreeze)inalighttightcontainer.

Ethanol

FormamideLowgrade(98%)forwashes(e.g.,Mallinkrodt3797)Highgradeforhybs(e.g.,BRL5515UA):Deionizemystirringwith5-15%(w/v)mixedbedresin(e.g.,BIORADAG501-X8,20-50mesh)for1h@RT.

Giemsa(SIGMAGS-500):Useata1:20dilutionin10mMNaHPO4,pH6.8.

2mg/mlGlycine(SIGMAG7126)in1XPBS

0.2NHCl

2MHEPES(SIGMAH3375,FW238.3)

Heptane

ImmersionOil(Fisher12-370A,CargilleTypeA)

KodakDeveloperD-19(cat1564598)Fixer(cat1971746)

Methanol

1MMgCl2(FWMgCl2-6H2O203.3)

1MMgSO4(FWMgSO4-7H2O246.5)

5MNaCl(FW58.44)

0.4%NaCl/0.3%TritonX-100

1MNaHPO4,pH6.869gNaH2PO4-H2O134gNa2HPO4-7H2OH2Oto1L

10NNaOH(FW40.0)

10XNTB0.5MTris-HCl,pH7.50.1MMgSO42.5mMDTT0.5mg/mlBSA

NTE0.5MNaCl10mMTris-HCl,pH81mMEDTA

Nucleotides3HdNTP35SrUTP3HrUTP,3HrCTP

O.C.T.(Tissue-TekO.C.T.Compound)

Paraffin(ParaplastPlus,Fisher)

8%Paraformaldehyde(PolysciencesE.M.Grade0380):Preparefreshin1XPBS.Dissolve8gparaformaldehydein100mlbufferat65-80degreesC.Add1-2drops50%KOHasnecessarybutbesurethefinalpHis7.5-8.AllowtocooltoRTbeforeusing.

10XPBS1.3MNaCl(58.44)=74.0gNaCl70mMNa2HPO4(142.0)=9.94ganhydrous30mMNaH2PO4(120.0)=3.6ganhydrous

Polylysine[SIGMAP1149(poly-D)orSIGMAP1399(poly-L),polylysinehydrobromidefraction150,000-300,000]:Either/orcomboofpoly-Dandpoly-Llysinemaybeusedtopreparea50-100ug/mlsolutionin10mMTris-HCl,pH8.

Polymount(Polysciences,Inc.)

Pronase(Calbiochem537088):Autodigesta40mg/mlstockinddH2Ofor4h@37degreesCtoinactivatetheRNase.Dispenseandfreeaein0.25mlaliquotsEachbatchshouldbetitratedforactivityinthepretreatmentprocedureuptothefixationstep(0.25-0.5mg/mlrangeiscommon).InsteadoffixingwithGiemsa,examinethesectionsandusethehighestcon-centrationwhichprovidesadequatepreservationoftissuemorphology.Pronasedilutionsshouldbemadein50mMTris-HCl,pH7.5/5mMEDTA.

Rubbercement(SanfordorCarter)

10XSalts3MNaCl0.1MTris-HCl,PH80.1MNaHPO4,pH6.850mMEDTA1XDenhardts

20XSSC-perliter:3MNaCl(58.44)=175.36gNaCl0.3MNa3-citrate(294.1)=88.24gNa3-citrate

STE0.8%NaCl20mMTris-HCl,pH820mMEDTA

StratageneTranscriptionKit

5XTB200mMTris-HCl,pH8250mMNaCl40mMMgCl210mMSpermidine

10%TCA(SIGMAT4885)

0.1MTriethanolamine-HCl,pH8(SIGMAT1502):Preparefresh,titratewithHCl.

1MTris-HCl,pH7.5127gTrizmaHCl47.2gTrizmaBaseH2Oto1L

1MTris-HCl,pH8.0177.6gTrizmaHCl106gTizmaBaseH2Oto1L

TritonX-100(SIGMAX-100)

WDTT1XSalts50%Formamide10mMDTT

Xylene