Immunofluroescence Technique-蚂蚁淘生物

Fixcellsin2%formaldehydeinPBS/pH7.4for15min.at20oC.2%formaldehydeismadeupfreshpriortousebydissolvingtheappropriateamountofEMgradeparaformaldehyde(Prillform,fromElectronMicroscopySciences)inPBSandheatingonahotplateinthehood(settingof5-6)untilthealdehydegoesintosolution.Keepthebottlecaploosenedsothatpressuredoesnotbuildup.Cooldownto20oCandpHto7.4.Alternatively,cellsmaybefixedin100%methanolat-20oCfor3minutes.Ifmethanolfixationisusedskiptostep4.

WashinPBS3X10min.

PermeABIlizein0.2%TritonX-100plus1%normalgoatserum(NGS)inPBS/pH7.3for5minutesonice.

WashinPBS+1%NGS3X10min.

Incubateintheappropriateconcentrationofprimaryantibodyfor1houratroomtemp.inahumidifiedchamber.Ifusing22mmX22mmsquarecoverslips,30ulofdilutedantibodyisplacedonthecoverslipandthecoverslipisinvertedontoaglassslide.Theslideisthenplacedinthehumidifiedchamberwhichisincubatedatroomtemp.

WashinPBS+1%NGS3X10min.

Incubateinsecondaryantibody(FITCorTexasRedconjugated)atadilutionof1:50for1hourinahumidifiedchamberatroomtemp.

WashinPBS4X10min.

Mountcoverslipwithadropofmountingmedium(see)andsealcoverslipwithclearnailpolishtopreventdryingandmovementunderthemicroscope.

From:Spector,D.L.andH.C.Smith.1986.Exp.CellRes.163,87-94.

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