Immunofluorescence staining of cells 免疫荧光染色Protocol
来自 : 蚂蚁淘
- Add100mlofwell-mixedanticoagulatedwholebloodtothebottomofalabeledtube.
- Addtheappropriateprimaryantibodytoeachtube.Ifusingunlabeledantibody,atitrationissuggested.Conjugatedantibodyshouldbeusedasdirected,usually20mlpersample.
- Mixwell,thenincubateinthedarkatroomtemperaturefor20-30minutes.
- Removetubesfromdarkchamberandmixeachtubewell.Add2mloflysingsolutiontoeachtube.Individuallyvortexeachtube.
- Incubateatroomtemperatureinthedarkfor10-15minutes.
- Centrifugefor5minutesat1000rpm(200xg).
- Removesupernatantbyaspiration,vortex,andadd2mlswashingsolutiontoeachtube.
- Centrifugefor5minutesat1000rpm(200xg).
- Removesupernatantbyaspiration.
- Ifrequired,addappropriatesecondstepantibody(atoptimalconcentration)toeachtubeandvortexgently(ifsecondstepantibodyisnotrequired,proceedtostep18).
- Incubateinthedarkatroomtemperaturefor20-30minutes.
- Removefromthedark.
- Mixwell,thenadd2mlwashingbuffertoeachtube.
- Centrifugefor5minutesat1000rpm(200xg).
- Removesupernatantbyaspirationandvortex.
- Ifthirdstepisnotrequired,proceedtostep18.Forthirdstep,addSav-PEtoeachtubeandvortexgently.
- Repeatsteps11-15.
- Ifyouwillanalyzethesameday,add500mlwashbuffertoeachtube,vortex,andanalyzewithin8hours.Ifnot,add2%formaldehydebuffertoeachtube,vortex,andstoreintherefrigeratorat2-8ºCforupto36hours.
Solutions:
WashingSolution:PBS+0.1%sodiumazide+1%fetalbovineserum.
Formaldehydebuffer:2%formaldehydeinPBS.
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