Step3-4:PermeABIlizewith1%TritonX-100inPBSfor10minutesatroomtemperatureandrinsewellwithPBS.

Steps5-6:Setuphumidifiedchamber(boxwithalidandwater-saturatedtissueinit;bottomlinedwithParafilm)andplacecoverslipsasdesired(e.g.touselowsolutionvolumes,intherangeof20-40ul,Pipetteantibodydirectlyontoparafilmandcarefullyplacecoverslipupsidedown,i.e.withcellsfacingdown,ontopofthedropofsolution;ifyou"reusingsolutionvolumesgreaterthan50ul,youcanplacethecoverslipontheparafilmwiththecellsfacingupandpipettetheantibodydirectlyontoit).

Beforeaddingantibody,however,blocknon-specificantibodybindingsitesbyincubatingcellsfor10mininblockingbuffer(PBS+0.1%Tween+1%serum).Afterthisstep,incubatecellswithprimaryantibodiesdilutedinblockingbufferfor35minsto1hour.Ifyouhaveneverusedtheantibodybefore,tryseveraldilutions(lowof1:100tohighof1:1000,forexample).

ClickheretoviewQuicktimemovieshowingtheset-upofthechamber(1.2Mb). ClickheretoviewQuicktimemovieshowingthetwodifferentmethodsofaddingantibodytothecoverslips(2.9Mb).

Clickonthumbnailtoviewfull-sizeimageshowingcoverslipssetupinahumidifiedchamber

Step7:Transfercoverslipsto6-wellplatesforPBSwashes(3x10min).Whenwashesaredone,transferbacktohumidifiedchamberforsecondaryantibodyincubation).Incubatecoverslipsfor35minsto1hourwiththeappropriatesecondaryantibodiesdilutedinblockingbuffer,andwash3X10minuteswithPBS.Weusefluorophore-conjugatedsecondaryantibodiesfromJacksonImmunochemicals.Transferbacktothe6-wellplateforfinalPBSwashes(3x10min),andthenstainwithDAPIifdesired(1:15,000dilutioninwaterofa5mg/mlstock).AgoodRNAstain,whichfluorescesintheredchannel,isPyroninY(1:10,000dilutioninwaterofa10mMstock;muststainwithDAPIfirst).WashwellwithPBS.

ClickheretoviewQuicktimemovieshowingthetransferofcoverslipstoa6-wellplateforPBSwashes(1.8Mb).

Clickonthumbnailtoviewfull-sizeimageshowingcoverslipsin6-wellplateforwashes

Step8:Mountthecellsforimmunofluorescenceanalysis.WecommonlyuseeitherMOWIOL/Dabco,whichhardens,ortheliquidmountingmediaVectashield(VectorLabs),inwhichcasethecoverslipsmustbesealedwithnailvarnish).Slidescanbestoredinthefridgeorfreezeruntilanalyzedbymicroscopy.

ClickheretoviewQuicktimemovieshowingthetwodifferentmethodsweusetomountcellsformicroscopy(5.7Mb).

Clickonthumbnailtoviewfull-sizeimageshowingthetwodifferentmethodsweusetomountcells

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