1.Fluorescentphalloidininmethanol.Phallacidindoesnotworkaswell.Dilute10ul330nMstockinto500ulPBSforeachlargecoverslip. 2.PBS,solutionA. 1.FixandpermeABIlizecells(seeotherprotocols).Mountcoverslipontoaplasticframereservedforfixedsamples. 2.Turnofflight.Dilutefluorescentphalloidin50xintoPBS. 3.Gentlypipetphalloidinsolutionontocoverslip.Stainfor30minatroomtemperature. 4.Rinsecoverslip3xwithPBS. 5.FillthechamberwithPBSoranantIBLeachingsolutionandobserve.Dishesmaybestoredat4oCinasealed,light-tightcontainer. 1.PBS/BSA:PBSsolutionAwith1%BSA(BoehringerMannheim100350)and0.1%NaN3,storedat4oC.Bringtoroomtemperaturebeforeuse. 2.Primaryantibody,dilutedappropriatelywithPBS/BSA.Need200ulper45x50mmcoverslip.ClarifyinaEppendorffor15min(minimalrequirement)orinanultracentrifugewiththeType42.2Tirotor(orAirfuge)ifnecessary. 3.Secondaryantibody,preparedasfortheprimaryantibody. 4.Coverslipboxes/containers. 1.Fixandpermeabilizecells(seeotherprotocols).WashwithPBS/BSAfor10mininafixationbox. 2.CutasmallpieceofParafilmtomatchtheareaofstainingandput200ulantibodysolutiononthepiece.Shakeoffmostoftheliquidfromthecoverslipbutdonotletitdryout.Invertthecoverslipontotheparafilm.Preparea100mmplasticpetridishcontainingapieceofwetfilterpaper.Place2woodensticksinthedishandputcoverslipupsidedownonthesticks.Sealthedishintoaziplockbagandplaceintheincubator.Stain45minat34-37oCwiththeprimaryantibody,orovernightat4oC. 3.Washgently3x,10mineach,withPBS/BSAonashaker.FillacoverslipboxwithPBS/BSAandsinkthecoversliptothebottom.Thecoveringparafilmshouldfloatup. 4.Stain30minwiththesecondaryantibodyasinstep2. 5.Washasinstep3. 6.Mountthecoverslipontoaplasticframereservedforfixedcoverslips.FillthechamberwithPBSoranantibleachingsolutionandobserve.Dishesmaybestoredat4oinasealed,light-tightcontainer.FLUORESCENTSTAININGOFCELLS
Materials
Procedure
Materials
Procedure(donotallowcoverslipstodryoutanytime)