Detection of Apoptosis in Paraffinembedded tissues by ISEL (In Situ End Labeling)相关交流-蚂蚁淘在线

Detection of Apoptosis in Paraffinembedded tissues by  ISEL (In Situ End Labeling)

蚂蚁淘 /发布时间:1630949408 /浏览量:322

DetectionofApoptosisinParaffin-embeddedtissuesbyISEL(InSituEndLabeling)

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.

Notes1)Inadditiontothehematoxylin,alighteosincounterstainmaybeusedtofurtheraidindetectionofapoptoticbodiesusingmorphologicalcriteria.ItisbesttousetheDAB+NichromagenifusinganH&Ecounterstain.

2)ThisprocedurecanbecoupledwithdetectionofBrdU.FollowtheISNTprocedureuptothecounterstainingstep(sectionE).ThenequilibrateslidesinautobufferandfollowtheBrdUstainingprocedurestartingatthe2NHClincubation.UseanAECchromagenfortheBrdUinsteadofDAB.Asectionofdoudenumisanexcellentpositivecontrolforthisdualstain.Thistissueshouldbestainedblackatthetipsofthevilli(ISEL)andredinthecrypts(BrdU).

================ 蚂蚁淘在线 ================

免责声明：本文仅代表作者个人观点，与本网无关。其创作性以及文中陈述文字和内容未经本站证实，对本文以及其中全部或者部分内容、文字的真实性、完整性、及时性本站不做任何保证或承诺，请读者仅作参考，并请自行核实相关内容

Detection&nbsp;of&nbsp;Apoptosis&nbsp;in&nbsp;Paraffinembedded&nbsp;tissues&nbsp;by&nbsp;&nbsp;ISEL&nbsp;(In&nbsp;Situ&nbsp;End&nbsp;Labeling)

Detection of Apoptosis in Paraffinembedded tissues by ISEL (In Situ End Labeling)