Immunofluorescence

1)GrowcellsinYEPDordefinedmediatoaconcentrationof1-5X107(protocolisfor~5mlofexponentiallygrowingcells).2)Add0.6mlof37%formaldehydesolutiondirectlytothecellsandincubatewithgentleshakingfor90minatthesametemperatureasgrowth.3)Transfercellstoa15mlconicaltubeandpelletbycentrifugation(3min@2000).4)Aspiratesupernatantandwashcellswith5ml0.1MKPO4,pH6.5.5)Repelletcellsandwashwith5ml0.1MKPO4,pH6.5/1.2Msorbitol(Psolution).6)Pelletcellsandaspiratesupernatant.ResUSPendin~1mlPsolution.7)Add25µlofDTTfor10minthen25µlofzymolyase.Incubatecellsontherollerat30°Cforonehour(oruntildigestionissufficient).8)Pelletcellsandaspiratesupernatant.Resuspendin~1mlofPsolution.9)Place20µlofcellsuspensionintoeachwellofapreparedslide.Afterafewminutesaspirateexcesscells.10)Immediatelyimmerseslideinice-coldmethanolfor6-7min.Removeandimmerseinice-coldacetonefor30s.Allowslidestoairdry.*Oncecellsarefixed-tryandkeepthewellsmoistatalltimes.11)Washslidesoncewith10mg/mlBSAin1XPBS.Removesupernatantandadd1°antibody(dilutedinBSA/PBS).Incubateinamoistchamberforatleast2h.12)Aspirateexcesssolutionandwash4XwithBSA/PBS.Add20µlof2°antibody(dilutedinBSA/PBS).Placeindark/moistchamberfor~2h.*Remembertokeepslidescoveredtopreventbleachingofthe2°Ab.13)Aspirateexcessandwash3XinBSA/PBS.Thenwashoncewith1XPBS.DiluteDAPI(1:1000)in1XPBS.AddDAPItowellsandallowtoincubateafewminutes.Aspirateexcess,washoncewith1XPBS,andallowslidestoairdry.14)Coverwellswithanti-fadereagentandcoverslip-sealwithclearnailpolish.

ImmunofluorescenceSolutions-

•0.1MKPO4,pH6.5•0.1MKPO4,pH6.5/1.2Msorbitol(Psolution)•zymolyase-makea10mg/mlsolutioninPsolution,vortexandletitsitonthebenchfor5min.Thenspininµfugefor5minanduseclearsupernatant•10mg/mlBSAin1XPBS•1XPBS•antifadesolution-p-phenylenediamine(1crystalin100µl10XPBSthenadd900µlglycerol)

SlidePreparation-Coatslidewellswith0.1%polylysine.Aspirateafter10-30s.Washeachwellwithwaterthreetimes.Allowtheslidetoairdryfor10min

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