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人类免疫缺陷病毒唾液抗体检测试剂盒怎么使用_康爱多网上药店

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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).

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