Materials

8%(w/v)paraformaldehydestocksolution:Dissolve8gofpowderedparaformaldehydein100mlwater.Heatandstir(55-60C–donotgohigher).Addafewdropsof2Nsodiumhydroxideuntilthesolutionclears.Makefresheachday.Alternatively,8%paraformaldehydecanbepurchasedfromElectronMicroscopySciencesasacustomformulationin4mlaliquots(cat.no.15710-SP).Throwawaywhateverisnotusedinoneday.

4%(v/v)paraformaldehyde:1:1solutionof8%paraformaldehydestocksolutionand0.2MPBS.Makeuponthedayofuse.

Microscopeslides:diptheslidesin1:10poly-l-lysinesolution(SigmaP8920)for2minutes.Allowtheslidestodry.

DNase(50U/ml)-ThestockisRQ1-RNAfreeDNase,Promegacat.#610A,concentration1U/µl.Dilute10µlDNasewith190µlPBS/PVP0µl.Usefresh.

RNase(50µg/ml)-UseRNaseA(heattreated),Qiagen,Germany,cat.#1901,concentration100mg/mlasastock.Dilute1µlRNaseA(stock)with999µlPBS/PVPandthendilutethissolution1:1withPBS/PVPtoobtainaworkingsolution(Usefresh).

Mountingmediumandantifade:ProLongAntifadeKit(MolecularprobesP-7481)

TUNELreactionmixture(InSituCellDeathDetectionKit,Fluorescein:BoehringerMannheim;Cat.No.1684795):Remove100µlLabelSolutionfrombottle2fortwonegativecontrols.Addtotalvolumeofbottle1(50µl)totheremaining450µlLabelSolutioninbottle2toobtain500µlTUNELreactionmixture.Mixwelltoequilibratecomponents.Note:TheTUNELreactionmixtureshouldbepreparedimmediatelybeforeuseandshouldnotbestored.

Hoescht33342:PrepareStock1bydissolving25mgHoechst33342(SigmaB2261)in2.5mlofdistilledwater(10mg/ml).Storeat4C.Onthedayofuse,prepareStock2bydiluting5µlStock1in10mlPBScontaining1mg/mlpolyvinylpyrrolidone(PBS-PVP)toproducea5µg/mlsolution.Theworkingsolutionispreparedbydiluting200µlStock2with800µlPBS-PVP(finalconcentration=1µg/ml).

Propidiumiodide(PI):Preparea2.5mg/mlstockbydissolvingPI(Sigma;catalognumberP4170)inPBS.Storethestockat4C.Immediatelybeforeuse,add100µlPIto900µlPBS/PVPandthenadd50µlofthisdilutedsolutionto200µlPBS/PVPtoobtainthefinalworkingconcentrationof50µg/ml.

Note:ToomuchPIcanobscuretheTUNELlabelingand,ifRNAisnotcompletelyremoved,leadtoexcessivecytoplasmicstaining.ItmaybenecessarytouselowerconcentrationsofPI,orshorterstainingtimetogetgoodresults.Wehaveusedconcentrationsaslowas0.5µg/mlPIwithgoodresults.IfpossIBLewerecommenduseofHoescht33342insteadofPI.

ProcedureforPerformingTUNELReactionforEmbryosinSolution(PreferredMethod)

1)Removeembryosfromembryoculturemedium(KSOM)andwash3timesin50µldropsofPBS-PVP(2minforeachwash)bytransferringtheembryosfromdroptodrop.

2)Fixembryosin50µldropsofparaformaldehydesolution[4%(w/v)inPBS,pH7.4]for1hatroomtemperature.Dropsmayevaporateifincubationiscontinuedforlongerthan1h.

3)Washtheembryos3timesina50µldropPBS/PVPbytransferringtheembryosfromdroptodrop(2minforeachwash).

4)Storetheembryosat4°Cin4-wellplatesuntiltheinitiationoftheTUNELprocedure(orproceedtoStep5withTUNELprocedure).Ifembryoswerestored,washtheembryosagainasdescribedinStep3.

5)Incubateembryosina50µldropofpermeABIlizationsolution[0.5%(v/v)TritonX-100,0.1%(w/v)sodiumcitrate]for30minatroomtemperatureinahumidifiedbox(aplasticboxwithwettowelswilldo).UseofaPAPpenorotherhydrophobicpenmayaidinformingthepermeabilizationdrop.Incubationinthepermeabilizationsolutiontoolongmaycauseembryostolyse.

6)FORPI:Prepareandlabel4dolphin-nosedtubes.OnecanaddPBS/PVPimmediatelybutdon’taddDNase,RNase,propidiumiodideorTUNELmixtureuntiljustbeforebeforeuse.

a.DNase(190µlPBS/PVP10µlDNase)

b.RNaseA(999µlPBS/PVP1µlRNase)

c.PreparePIasdescribedabove.

d.TUNEL(emptyfornow)

7)WashtheembryosasdescribedinStep3.ForpositiveandnegativecontrolsgotoStep8.Forsamples,proceedtoStep9.

8)Forpositiveandnegativecontrolembryosonly-IncubatepositiveandnegativecontrolembryoswithRNAfreeDNase(50U/ml)at37°Cfor1hinthedark.WashembryosasdescribedinStep3.(ContinuetoStep9a).

9)Incubateembryosin25µldropsoftheTUNELreactionmixturefor1hourat37°Cinthedark(placeembryosinboxcoveredwithaluminumfoil).

a.Forpositiveandnegativecontrolembryosonly-IncubatepositivecontrolembryosasdescribedinStep9.Incubatenegativecontrolsintheabsenceoftheenzymeterminaltransferase(bottle1),onlywithlabelsolutionfrombottle2.ContinuetoStep10.

10)WashtheslidesasdescribedinStep3.

11)IfusingPI,incubateembryoswithRNaseA(50µg/ml)for1hatroomtemperatureinthedark.ThisstepcanbeomittedifHoescht33342isusedtostainnuclei.

12)Incubateembryosina25-50µldropofHoescht33342orPIfor15minatroomtemperatureinthedark.

13)Washtheembryos7-8timesinPBS/PVPasdescribedinStep3.

14)Add2-3µlofmountingmedium(w/antifade)toapoly-L-lysinecoatedslide.

15)Transfertheembryos(5to20embryosperdrop)andcoverthesamplewithacoverslip.

16)Determinethenumberoffluorescentnuclei.Blue(Hoescht)orred(PI)fluorescenceindicatesnon-apoptoticcellsandgreenish-blueorteal(Hoescht)orgreen/yellow(PI)fluorescenceindicatesapoptoticcells.

HINT:Ifbackgroundfluorescenceisaproblem,washtheembryosforalongerdurationinStep13.Itiscrucialthatthenon-specificPIiswashedawayfromembryospriortomountingthem.

ProcedureforPerformingTUNELReactionforEmbryosAffixedtoMicroscopeSlides

NOTE:TheprocedureiswrittenforPIbutcanalsobeusedwithHoeschtinstead

1)Removeembryosfromembryoculturemedium(KSOM)andwash4timesin100µldropofPBS1mg/mlpolyvinyl-pyrrolidone(PVP)bytransferingtheembryosfromdroptodrop.

2)Fixembryosin100µldropofparaformaldehydesolution[4%(w/v)inPBS,pH7.4]for1hatroomtemperature.Washtheembryos3timesin100µldropPBS/PVPbytransferingtheembryosfromdroptodrop.Transfertheembryostoapoly-l-lysinecoatedslideandallowembryostodryfor24hoursatroomtemperature.

3)WashtheslidestwicebydippinginaCoplinjarcontainingPBS/PVP(2minuteseach).

4)Incubateinpermeabilisationsolution[0.5%(v/v)TritonX-100,0.1%(w/v)sodiumcitrate]for30minatroomtemperature.IFyougotoolong,theembryomaylyse.

5)Washtheslidesasdescribedinstep3.

6)IncubatepositiveandnegativecontrolwithDNase(50U/ml)at37Cfor1h.

7)Washtheslidesasdescribedinstep3.

8)Drytheareaaroundthesampleandadd50µlofTUNELreactionmixture.Incubatefor1hourat37Cinthedark.Incubatenegativecontrolsintheabsenceoftheenzymeterminaltransferase(labelsolutionfrombottle2).

9)Washtheslidesasdescribedinstep3.

10)IncubatetheslideswithRNaseA(50µg/ml)for1hatroomtemperature.

11)Blottheslides,drytheareaaroundthesampleandaddpropidiumiodide(0.5µg/ml)for1hatroomtemperature.

12)Washtheslides4timesinPBS/PVP(2minuteseach).

13)Drytheareaaroundthesample,add16µlofmountingmedium(w/antifade)totheslideandcoverthesamplewithcoverslip.

14)Determinethenumberoffluorescentnuclei.Redfluorescenceindicatesnonapoptoticcellsandgreen/yellow(PI)or(Hoescht)fluorescenceindicatesapoptoticcells.

Confocalimageofabovineembryoheatshockedat41Cfor9handsubjectedtotheTUNELprocedureusingPI.

Epifluorescentimageofabovineembryoheatshockedat41Cfor9handsubjectedtotheTUNELprocedureusingHoescht33342.

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• 活性染料和化合物
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