Programmed Cell Death In Situ Detection Using Terminal deoxynucleotidyl Transferase (TdT)相关交流-蚂蚁淘在线

Programmed Cell Death In Situ Detection Using Terminal deoxynucleotidyl Transferase (TdT)

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ProgrammedCellDeathInSituDetectionUsingTerminaldeoxynucleotidylTransferase(TdT)

TerminaldeoxynucleotidylTransferase-mediateddUTPnickendlabeling(TUNEL)isaninsitumethodfordetectingthe3"-OHendsofDNAexposedduringtheinternucleosomalcleavagethatoccursduringapoptosis.IncorporationofbiotinylateddUTPallowsdetectionbyimmunohistochemicalprocedures.Thelabeledapoptoticcellsmaybevisualizedbylightmicroscopy.

Reference:Gavrielietal.,J.ofCellularBio.119:493-501,1992.

DEPARAFFINIZE1.Heatto70^oCfor10minorto60^oCfor30min.2.Immediatelyplaceslidesinxylene2x5min96%EtOh2x3min90%EtOh1x3min80%EtOh1x3mindiH₂O1x3min3.CirclesectionswithPAPpenandreturntodiH₂O.

PRETREAT1.IncubatewithProteinaseKatRTfor30min.2.WashwithdiH2O,4x2min.3.Incubatewith2%H₂O₂atRTfor10min.4.RinsewithdiH₂O.

HYBRIDIZE1.CoverslideswithTdTbuffer,tapoff.2.AddTdT/dUTPsolution.3.Incubateinhumidchamberat37^oCfor1hour.

POSTHYBRIDIZATION1.SubmergeslidesinTBbufferatRTfor15min.2.RinseindiH₂O.3.Coverslideswith2%BSAatRTfor10min.4.RinseindiH₂O.5.ImmerseslidesinPBSatRTfor5min.

DETECTION1.IncubatewithdilutedExtraavidin-peroxidaselinkfor30minat37^oC.2.WashwellwithastreamofdiH₂O.3.ImmerseinPBS,blotoff.4.MixAECsubstratesandaddtoslide.5.DevelopcolouratRTtodesiredintensity,approximately3min.6.RinsewithdiH₂O.7.CounterstainwithmodifiedHarris"hematoxylinandbluewithPBS.

COVERSLIP1.Airdry,thenmountwithCrystalMount.2.Bakeat65^oCfor15-45min.

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

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Programmed&nbsp;Cell&nbsp;Death&nbsp;In&nbsp;Situ&nbsp;Detection&nbsp;Using&nbsp;Terminal&nbsp;deoxynucleotidyl&nbsp;Transferase&nbsp;(TdT)

Programmed Cell Death In Situ Detection Using Terminal deoxynucleotidyl Transferase (TdT)