TheISELtechniqueidentifiesmorphologicallyapparentkaryorrheticcellsandalsolabelscellsinwhichtheDNAfragmentationhasnotyetprogressedtofranknuclearfragmentation.ByusingbiotinylatednucleotidesandaDNApolymerase,theDNAfragmentsserveastemplatesinsitutosynthesizenewDNAstrands,whicharevisualizedbylightmicroscopyafterhistochemicalprocessing. Reference:Wijsmanetal.,JofHistochem.Cytochem.41(1):7-12,1993. PROCEDURE Todeterminetheoptimalconditionsforeachstudy,performthefollowing“titration”usingthestandardprotocolbelow.Serialsectionsfromthesameblockprovidethebestsamplesforthetitration. SAMPLEdUTPdilutionPOLIdilutionDIGESTION1A1:1001:200NONE2A1:2501:200NONE3A1:5001:200NONE4A1:1001:500NONE5A1:2501:500NONE6A1:5001:500NONE 1B1:1001:200TRYPSIN2B1:2501:200TRYPSIN3B1:5001:200TRYPSIN4B1:1001:500TRYPSIN5B1:2501:500TRYPSIN6B1:5001:500TRYPSIN 1C1:1001:200PEPSIN2C1:2501:200PEPSIN3C1:5001:200PEPSIN4C1:1001:500PEPSIN5C1:2501:500PEPSIN6C1:5001:500PEPSIN A.DEPARAFFINIZEANDHYDRATE5mineachin:xyleneX2xylene:EtOH(1:1)100%EtOH90%EtOH70%EtOHdiH2OX2 B.PRETREATBlockendogenousperoxidasebyimmersingslidesin3%H2O2for30minatRT.Prepare2XSSCandpreheatto80°C.RinseslidesindiH2O.Washin0.15MPBS3X4minIncubateinthepreheated2XSSCat80°Cfor20min.Rinseoncein0.15MPBS,thenwashin0.15MPBS3X4min.Ifnecessaryasdeterminedbytitration,incubateslidesunderdesiredenzymedigestionconditions.Rinseoncein0.15MPBS,thenwashin0.15MPBS3X4min.EquilibrateinBufferA3X4min. C.LABELPrepareISELsolution,addingenzymeimmediatelybeforeuse.IncubateslidesinISELsolutionin18°Cicebathfor2hrs.PrepareABCsolutionnearendofISELincubation,foruse30to60minuteslater.RinseinBufferA,thenwashinBufferA3X4min.Washin0.5MPBS3X4min. D.DETECTApplyABCsolutionandincubateatRTfor30min.Washin0.5MPBS3X10min.PrepareDAB+Ni.ApplyDABfor3to10minatRT.CheckreductionofDABundermicroscope.Whencolorhasdevelopedtothedesiredintensity,rinseinrunningdiH2Ofor2minutes. E.COUNTERSTAINImmerseinHematoxylinfor90sec.Rinseinwater.BlueinLithiumchloridefor20sec.Rinseinwater.Rehydrateandcoverslip. 2)ThisprocedurecanbecoupledwithdetectionofBrdU.FollowtheISNTprocedureuptothecounterstainingstep(sectionE).ThenequilibrateslidesinautobufferandfollowtheBrdUstainingprocedurestartingatthe2NHClincubation.UseanAECchromagenfortheBrdUinsteadofDAB.Asectionofdoudenumisanexcellentpositivecontrolforthisdualstain.Thistissueshouldbestainedblackatthetipsofthevilli(ISEL)andredinthecrypts(BrdU).
Notes1)Inadditiontothehematoxylin,alighteosincounterstainmaybeusedtofurtheraidindetectionofapoptoticbodiesusingmorphologicalcriteria.ItisbesttousetheDAB+NichromagenifusinganH&Ecounterstain.
