ProgrammedCellDeathInSituDetectionUsingTerminaldeoxynucleotidylTransferase(TdT) TerminaldeoxynucleotidylTransferase-mediateddUTPnickendlabeling(TUNEL)isaninsitumethodfordetectingthe3"-OHendsofDNAexposedduringtheinternucleosomalcleavagethatoccursduringapoptosis.IncorporationofbiotinylateddUTPallowsdetectionbyimmunohistochemicalprocedures.Thelabeledapoptoticcellsmaybevisualizedbylightmicroscopy. Reference:Gavrielietal.,J.ofCellularBio.119:493-501,1992. DEPARAFFINIZE1.Heatto70oCfor10minorto60oCfor30min.2.Immediatelyplaceslidesinxylene2x5min96%EtOh2x3min90%EtOh1x3min80%EtOh1x3mindiH2O1x3min3.CirclesectionswithPAPpenandreturntodiH2O. PRETREAT1.IncubatewithProteinaseKatRTfor30min.2.WashwithdiH2O,4x2min.3.Incubatewith2%H2O2atRTfor10min.4.RinsewithdiH2O. HYBRIDIZE1.CoverslideswithTdTbuffer,tapoff.2.AddTdT/dUTPsolution.3.Incubateinhumidchamberat37oCfor1hour. POSTHYBRIDIZATION1.SubmergeslidesinTBbufferatRTfor15min.2.RinseindiH2O.3.Coverslideswith2%BSAatRTfor10min.4.RinseindiH2O.5.ImmerseslidesinPBSatRTfor5min. DETECTION1.IncubatewithdilutedExtraavidin-peroxidaselinkfor30minat37oC.2.WashwellwithastreamofdiH2O.3.ImmerseinPBS,blotoff.4.MixAECsubstratesandaddtoslide.5.DevelopcolouratRTtodesiredintensity,approximately3min.6.RinsewithdiH2O.7.CounterstainwithmodifiedHarris"hematoxylinandbluewithPBS. COVERSLIP1.Airdry,thenmountwithCrystalMount.2.Bakeat65oCfor15-45min. SOLUTIONS ProteinaseK,20ug/mlDilute1.35D14.8mg/mlSTOCKin999DPKbuffer 2%H2O2Dilute6.67ml30%STOCKin100mldiH2O 2%BSADissolve2gofBSAin100mldiH2O TdT/dUTPsolution,(1:50/1:10)prepare75Dperslide:1.5DTdT7.5DdUTPin66DofTdTbuffer Extraavidin-Peroxidase(1:10)prepare100Dperslide10Din90DofdiH2O AECsubstrateTo5mlH2O,add2dropsA3dropsB2dropsC,mixwell. STOCKSOLUTIONS TdTBuffer,100ml30mMTris,pH7.23mlof1M140mMNaCacodylate2.24g1mMcobaltchloride1mlof100mM 100mMCobaltchloride,100ml2.379g 10XTBBuffer,100ml3MNaCl17.53g0.3MNacitrate8.823gdiluteto1xbeforeuse. ProteinaseKbuffer,100ml50mMTris(8.0)5mlof1Mstock1mMEDTA200ulof0.5Mstock PRODUCTSANDSUPPLIERS ProteinaseKsolutionBoehringerManneheim:1413-783 Biotin-16-dUTPBoehringerManneheim:1093-070 TerminaltransferaseGibco/BRL:8008SB(TdT) ExtraAvidinPeroxidaseSIGMA:E2886 AECSubstratekitVector:SK-4200 HarelcoHarrishemotoxylinBaxter:57735-3 CrystalmountBioMeda:M02
