TISSUE PREPARATION FOR IN SITU HYBRIDIZATION

1. HarvesttissueandrinseinPBSorsaline.
2. Immersetissuein4%paraformaldehyde/0.1MsodiumphosphatebufferpH7.4(recipefollows)at4°Cfor1-3hrs.Trytoavoidovernightfixationifpossibleasthiscausesproblemswithtissueadherenceonslidesduringthehybridizationprocedure.
3. Immersetissueinsterile15%sucrose/1xPBS(recipefollows)3hrs.toovernightat4°C.
4. EmbedtissueinO.C.T.(BaxterNo.M7148-4),M1(Lipshaw)oranyotherconvenientembeddingmatrixforfrozensectioninginplasticembeddingmolds(.Tissueshouldbeorientedintheblockappropriatelyforsectioning(cross-section,longitudinaletc.).Notethetissuenumberontheblockdirectlyforreference.
5. Freezetissueblockinliquidnitrogen.Placethebottomthird(aproximately)oftheblockintotheliquidnitrogen,allowtofreezeuntilallbutthecenteroftheO.C.T.isfrozen,andallowfreezingtoconcludeondryice.
6. Storetissueblocksat-70°Cinasealedcontainerorwrappedinfoilandshipondryice.

Itisalsopossibletousefreshfrozentissueforinsituhybridizationiftheparaformaldehyde/sucrosemethodisnotfeasible.TissuesshouldberinsedinsalineorPBSandfrozeninO.C.T.blocksinliquidnitrogenasoutlinedabove.Althoughnotoptimal,itisalsopossibletousesnapfrozenmaterialtissuewithoutanembeddingmatrix.Thefixation,sucrose,andO.C.T.stepsareusedprimarilytoimprovethetissuemorphology.

Itisexpectedthatthefixationtimesoutlinedabovewillnotresultincompletefixationoflargepiecesoftissue.However,theadditionalfixationstepatthebeginningoftheinsituhybridizationprocedureshouldensureadequatefixationofsuchtissuespriortohybridization.

Thisprotocolhasbeenusedsuccessfullyonlarge(upto1cubiccm)andsmall(1cubicmm)tissuesamples.

4%Paraformaldehyde

Mixinatwoliterflask:

200ml0.5MNaPO4,pH7.4800mldepcH20Heatto70°CwithstirringonhotplateinfumehoodAdd40gParaformaldehyde(EMgrade,Polysciences,CatNo.0380)

Oncethesolutionhascleared(itshouldtake5minutesorless),filterwithaside-armflask,BuchnerfunnelandWhatmanNo.2filterpaper.Immediatelypourthesolutionintoaoneliterbottlewhichhasbeenpackedinice.Thiscoolsthesolutionquicklyandpreventsbreakdownoftheparaformaldehyde.Storeat4°Cforuptotwoweeks.

15%SucroseinPBS:

500mlsterilePBS75g"RNasefree"sucrose

MixaboveandfiltersterilizewithadisposableNalgenefiltrationunittypeS(0.45micron).Storeat4°C.

USEOFFISHERBRANDSUPERFROST/PLUSMICROSCOPESLIDESFORINSITUHYBRIDIZATION

WeuseFisherbrandSuperFrost/Pluspositively-chargedmicroscopeslides(Cat.No.12-550-15)forallofourfrozentissuesectioningandhaveverygoodtissueretentiononslidesafteraninsituhybridizationexperiment.SuperFrost/Plusslidesrequirenopreparationtimepriortocryosectioningandarecompetitiveintermsoflaborcostandreagentexpenses.

SECTIONINGOFFROZENTISSUESFORINSITUHYBRIDIZATION

1. Frozentissuespreparedasdescribedcanbewrappedandstoredformanyyearspriortosectioning,withoutlossofthemRNAsignal.ThebiggestproblemwithstoredtissueblocksisthattheytendtodessicateifnotproperlywrappedandtheO.C.T.(OptimalCuttingTemperaturecompound)canbedifficulttocut.
2. Blocksshouldberemovedfromthe-70°Cfreezerandallowedtoequilabratewiththecryostatchambertemperature.Tissuescanbecutatanyconvenienttemperature(-15to-35°C)asneeded.Mosttissuescutwellat-15°C(brain,kidney,liver,vessels,muscle,etc.)howeverfattyormoredifficulttissues(adiposetissue,skin,lung)requiretemperaturesaslowas-35°Corlesstoobtaingoodsections.Careshouldbetakennottotouchthefaceoftheslidesbuthandlebytheedgesonly.Frozensections5-7µm(thinnerisOKbutthicker,over10µm,maypresentproblemsforvisualizingmRNAinsitu)shouldbecut,thaw-mountedontoroom-temperatureslides,andimmediatelyrefrozenbyplacingslideswithsectionsintoaslidebox(VWRmicroslidebox#48444-003)withasingledessicantcapsule(Humi-Capseebelow).Whentheboxisfull,placethetopontheboxandstoreat-70°C.Sectionscutandstoredwithdessicantarestableforinsituhybridizationandimmunohistochemistryformostantigensforover5years.