In Situ-丁玉强

download In Situ-丁玉强

of 23

  • date post

    13-Nov-2014
  • Category

    Documents

  • view

    113
  • download

    1

Embed Size (px)

Transcript of In Situ-丁玉强

1

AndersonLabInSituHybridizationProtocolsMarch1997

These protocols describe nonradioactivemethods for in situ hybridization on frozen sections, whole mount embryosandonculturedcells.They havebeenfreely adapted and modified from the protocols of Richard Harland, David Wilkinson, DomingosHenrique,AndyMcMahon,TonyCampagnioni,andothers.Thisprotocolis modifiedfromtheNovember1995versionpreviouslycirculated.

2

I:InSituHybridizationofFrozenSectionsSectionsarecollectedonSuperfrost/PlusslidespurchasedfromFisheranddried inairforonehouratroomtemperature,thenstoredat20C.Alternatively,useRNAse freeslidescoatedwithTESTA,inwhichcaseyoumustdrytwohours.Itissometimes necessarytowashtheslidesinDEPCPBSandthreechangesofDEPCwaterbefore storingat20C.Thisdependsonboththeprobeandembeddingmaterialused.Details ofhowtoprepareTESTAcoatedslidesareintheAppendix. A:PreTreatmentofSections N.B.AllreagentsandmailersinthefollowingstepsmustbeRNAsefree. Inthissection,steps6and7(theacetylation)isvitalforsomeprobes,butraisesthe backgroundforothers.Someprobes,likeSCG10,Brn3andneurofilament,work bestwithoutacetylation.Otherprobes,likeGATA2,requireit.Someworkokay withoutitbuthaveimprovedsignaltobackgroundifacetylationisincluded.I recommendcomparingsisterslideswithandwithoutacetylationthefirsttimeyou tryaprobe. 1.Warmslidestoroomtemperatureanddryat50Cfor15minutes. 2.Fixin4%paraformaldehydeinDEPCPBSatroomtemperaturefor20minutes. 3.WashtwiceinDEPCPBSatroomtemperaturefor5minutes. 4.Treatslideswith50g/mlProteinaseKinPKbufferatroomtemperatureforbetween 815minutesdependingontheageoftheembryo. 5.WashonceinDEPCPBSatroomtemperatureforfiveminutes.Fixin4% paraformaldehydeinDEPCPBSfor15minutes. 6.RinseonceinDEPCwater. 7.PlaceslidesinanRNAsefreeglasstroughwithastirbar.Add250ml0.1MRNAse freetriethanolamineHClpH8.0.Add0.625mlaceticanhydride(CARE!)withconstant stirring.Turnoffstirrerwhentheaceticanhydrideisdispersedandleaveforafurther10 minutes. 8.WashslidesinDEPCPBSatroomtemperatureforfiveminutes. 9.Prehybridisefor34hoursat60C.Replacewith12g/mlofprobeandcontinue incubationforafurther1216hours.Thesestepsshouldbeperformedinthe

3 hybridizationoven,nottheregular60Coven,asthelatterdoesnotmaintainits temperaturewell. N.B.Wehavefoundthatthemosteffectivewaytocarryoutthehybridisationisinslide mailers.Itisagoodideatothoroughlysealthelidsoftheslidemailerswithparafilmto preventevaporationofprobe. B:WashingSteps N.B.Afterhybridisation,itisnotnecessarytouseRNAsefreebuffers. 1.Placeslidesinatroughwithastirbar.Washin1xSSCat60Cfor10minutes. 2.Washin1.5xSSCat60Cfor10minutes.Coolslidesto37C. 3.Washtwicein2xSSCat37Cfortwentyminuteseach. 4.Treatwith0.1g/mlRNAseAin2xSSCat37Cfor30minutes. 5.Washin2xSSCatroomtemperaturefor10minutes. 6.Washtwicein0.2xSSCat60Cfor30minuteseach. 7.Washoncein0.2xSSCatroomtemperaturefor15minutes. 8.WashonceinPBTfor15minutes. 9.Incubateslidesin20%heatinactivatedsheepseruminPBTforbetweenoneandfive hoursatroomtemperature.Forsomeprobes,thelongerincubationseemstocutdown onbackground. C:AntibodyVisualisationofDigoxygenin 1.Incubateslideswithpreabsorbedantidigoxygeninantibody(coupledtoalkaline phosphatase)dilutedtoafinalconcentrationof1:2000in20%sheepseruminPBTat 4Covernight. 2.WashthreetimesinPBTatroomtemperaturefor30minuteseach. 3.WashtwiceinAlkalinePhosphatasebufferatroomtemperaturefor5minuteseach. Levamisoleissometimesincludedinthesecondwashandreactionbufferasan inhibitorofendogenousalkalinephosphatase,butonmostembryosectionsthe endogenousactivitydoesnotsurvivethehybridizationprocedure. 4.ForeverymlofAlkalinePhosphatasebuffer,add1lofNBTand3.5lofBCIP,and developinthedarkforbetween220hours,dependingontheabundanceoftheRNA. Sincethealkalinephosphataseenzymeisverystable,itispossibletowashoutthe NBT/BCIP,replacewithalkalinephosphatasebuffer,andtocontinuethereactionata latertime.

4 5.WashtwiceinPBStoremovesubstrates. 6.FixslidesinMEMFAforatleast15minutesatroomtemperature.Mountslidesin glycerol/PBS. N.B.IftheBCIP/NBTprecipitateisnotfixed,itwillslowlydarkenoverthecourseof aboutamonth.Eventually,thebackgroundwillbecometoodark.

AdditionalProtocolsforsections:Doublelabelprotocols.ThesemodificationswerecontributedbyTetsuchiroSaito. Modificationsfortwoprobedoublelabelprotocol Thisprotocolworksbestfordemonstratingtwopopulationsofcellsexpressingtwo separatemarkers,sincebothproductsarecytoplasmic,andtheNBT/BCIPproduct (purple)canobscuretheINT/BCIPproduct(red). A:Preparationofprobes. 1.AprobeispreparedbyusingfluoresceinRNAlabelingmixinplaceofdigoxygenin NTPs. B:Hybridization. Boththedigoxygeninlabeledprobeandthefluoresceinlabeledprobeareincludedin thehybridizationbufferat12g/mleach. C:Staining. Thefollowingstepsareperformedinslidemailers. 1.SectionsareincubatedwitheitherantifluoresceinAPFabfragmentsoranti digoxygeninAPFab,preabsorbedandattheappropriatedilutionin20%sheepserum inPBT,at4Covernight.Staintheweakerprobefirst. 2.Afterwashing,stainwithNBT/BCIPasinthenormalprotocol,untilstainingreachs thedesiredintensity.

5 3.WashthreetimesinPBSatroomtemperaturefor5minuteseachtoremovesubstrates. 4.IncubateinTE(100mMTrisHClpH7.5,50mMEDTA)at85Cfor10minutesto inactivatealkphos.Usethe68Cwaterbathturnedupto85C. 5.WashthreetimesinPBSatroomtemperaturefor5minuteseach. 6.WashonceinPBTatroomtemperaturefor10minutes. 7.Incubateslidesin20%heatinactivatedsheepseruminPBTatroomtemperaturefor 30minutes. 8.Incubateslideswiththeotherantibodyat4Covernight.Ifyouusedantifluorescein originally,useantidigoxygeninastheotherantibody,andviceversa. 9.WashthreetimesinPBTatroomtemperaturefor30minuteseach. 10.WashtwiceinAlkalinePhosphatasebufferatroomtemperaturefor5minuteseach. 11.ForeverymlofAlkalinePhosphatasebuffer,add7.5lINT/BCIPstocksolution,and developinthedarkforbetween220hours,dependingontheabundanceoftheRNA. N.B.AntidigoxygeninAPFabfinalconcentrationis1:2000.AntifluoresceinAPisused at1:4000.

Modificationsforoneprobe,oneantibodydoublelabelprotocol Sinceeachantibodyepitopecanvaryinstability,thisprotocolmustbespecifically tailoredfortheantibodyused.Tetsuchirodoesnotrecommendtryingmembraneor cytoplasmicantigensfordoublelabel;nuclearantigensbestsurvivethehybridization procedure.ThisistheprotocolIhaveusedforcombiningthequailnuclearantigenwith insituhybridization.(PMW) A:PreTreatmentofSections TestdifferentproteinaseKconcentrationstofindwhichprovidesthebestcompromise betweeninsitusignalandantibodysignal.Forexample, 100%proteinaseK 50g/mL 10%proteinaseK 5g/mL 1%proteinaseK 0.5g/mL substitutedintothenormalhybridizationprocedure.Alsotrythefollowing: 1.Warmslidestoroomtemperatureanddryat50Cfor15minutes.

6 2.Fixin4%paraformaldehydeinDEPCPBSatroomtemperaturefor20minutes. 3.WashtwiceinDEPCPBSatroomtemperaturefor5minutes. 4.Prehybridisefor34hoursat60C.Replacewith12g/mlofprobeandcontinue incubationforafurther1216hours.

B:WashingSteps Followthenormalprotocolforstringencywashesthroughthe15minutewashwithPBT (steps18). 9.Blockwith1%goatserum,1%BSA,0.5%TritonintissueculturegradeDPBSatroom temperaturefor1hour. 10.Incubateovernightwith1:1QCPNsupernatantand1%goatserum,1%BSA,0.2% TritoninTCDPBS. C:HRPDevelopment 1.WashthreetimesinPBTatroomtemperaturefor5minuteseach. 2.WashthreetimesinPBSatroomtemperaturefor5minuteseach. 3.Inactivatetheendogenousperoxidasewith0.3%H2O2inmethanolatroom temperaturefor30minutes.Ihavetestedslidespretreatedwith1g/mLproteinaseK andhybridizedat60Cfor18hours,followedbynormalSSCwashes,andhavefound residualendogenousperoxidaseactivityintheredbloodcells. 4.WashthreetimesinPBSatroomtemperaturefor5minuteseach. 5.WashthreetimesinPBTatroomtemperaturefor5minuteseach. 6.Incubatewithsecondaryantibody(goatantimouseHRP,inthiscase)in1%goat serum,1%BSA,0.1%TritoninTCDPBS. 7.WashthreetimesinPBTatroomtemperaturefor5minuteseach. 8.WashonceinPBTat4Covernighttofurtherreducebackground,ifnecessary. 9.Washthreetimesinacetateimidazolebufferatroomtemperaturefor5minuteseach. 10.DevelopHRPreactionwithnickelDABinacetateimidazolebuffer. 11.WashthreetimesinPBSatroomtemperaturefor5minuteseach. 12.WashonceinPBTatroomtemperaturefor10minutes. 13.Incubateslidesin20%heatinactivatedsheepseruminPBTatroomtemperaturefor onehour. Nowfollowthenormalprotocolforvisuallizationofdigoxygenin(orfluorescein.)

7 Veryimportant:FixingtheslidesinMEMFAeliminatestheNiDABsignal!Mountand viewtheslidesrightaway.

Troubleshootingandcommonproblems. A.Lowsignal/highbackground Isyourovenmaintainingtemperature?Arepeoplegoinginandoutofit?Ifyoure usingahybridizationoven,youcanalsoconsidertrying68Cor72C. Ifyouarereusingthe20%sheepserumblockorantidigoxygeninantibody,checkitfor particulates,orjustreplaceit.(especiallyforspottybackground) Beextremelyrigorousabouthowlongyourslidesarein0.2XSSC.Thishighstringency stepstripstheprobeoffthesections.Itisnecessarytoreducebackground,butIhave foundthatwithcleanprobes,thehighstringencywashescanbeparedbackto25 minutesandthisimprovessignal.ThesameistruefortheRNAseAstep.(PMW) Didyouhydrolyzeyourprobe?Poorsignalisalsocommoniftheprobeislessthan500 bp.(Hairarelyhydrolyzeshisprobes,though,andhiswholemountsarelovely.) Checkforparticulatesinthephosphatebufferusedinmakingthefixativeorsucrosefor youranimals.ContaminatedPBusedtomakeeitherreagentcausesunholybackground forantibodystaining,andseverelyreducessignalforinsitu. B.Unevenlabelingacrosstheshortaxisoftheslide. Thereissomedebateastowhatcausesthisphenomenon.Andysaystoreducethe speedofthestirbarduringtheSSCwashestounder3.Patsaysitsevaporationof formamideduringhybridization,andtousefreshprobenexttime.Tryboth.

8

II:WholeMountInSituHybridization.Embryosshouldbedissectedfreeofanyextraembryonicmembranes,fixedin 4%paraformaldehydeinDEPCPBSfor2hoursatroomtemperature(or4C overnight),washedinDEPCPBS,andthenstoredin100%methanolat20Cuntil required.Haipuncturestheembryosontheforeheadandhindbrainregionwithafine needletoallowfreeexchangeofreagents. Ingeneral,wedothewashesandincubationsin15mltubescontainingabout56 mlliquid.Thetubesarerockedgentlyonarotatingplatformtoallowthorough exch