1)Immerse18mm2glasscoverslipsinEtOH.Inthetissueculturehood,individuallypulloutandcarefullyflametosterilize.Allowtocoolthenplacein35mmdishes,orin6wellplates.Coatovernight(4°C)with3ml/dishorwellof40µg/mlBMSmadeupinddH2O. 2)Removecoatingsolutionandblockwith1%BSAfor4hrat4°C. 3)Isolatecellsandplateat6x105cells/ml(3ml/well).Allowcellstoadherefor1hrinincubator.Gentlypulloffmediacontainingnon-adherentcellsandwashoncewithmedia.Pulloffmediaandadd4%formaldehyde(25mlof16%ampulestockmadeupto100mlinPBS)todishesorwells.Fixfor30minatroomtemperature. 4)Removecoverslips.Thoseforimmunostainingareplacedverticallyintwoceramiccoverslipholders.Theremaindercanbestoredat-70°C. 5)CoverslipsinceramicholdersarewashedtwotimesinPBS.Forwashesandincubations,usea250mlbeakeranda100mlvolumeofwashorreagent.PermeABIlizebyimmersioninPBS-Tween("PBS-T";PBScontaining0.1%Tween20)for15minatroomtemperature.Washfourtimesover5minwithPBS. 6)BlockbyimmersioninPBScontaining1%BSAfor60minatroomtemperature.ImmerseonegroupofcoverslipsinpreimmuneseraandtheotherinimmuneseradilutedinPBS/1%BSA.Coverandincubateovernightat4°C. 7)Thenextday,washfivetimesover40mininPBS.Atthistime,canimmerseinPierce"peroxidasesuppressor"for30minatroomtemperature,thenwashseveraltimesinPBS.Immerseinsecondaryperoxidase-labeledantibodydiluted1/1,000inPBScontaining1%normalgoatserum.Incubatefor60minatroomtemp.Washfivetimesover40mininPBS. 5)PlacecoverslipsflatonParafilmandaddPierce"metalenhancedDAB"diluted1/10inperoxidebuffer.Allowreactiontogofor5-15min,thenreplaceinceramicholderstowashtwotimesinwater.Dehydratein80%,90%and2x100%EtOH,thenimmerseinxylene(inchemicalhood;all1mineach).Placecoverslipcelllayerdownonaglassslidecontainingadropofmountingmediumandexamineinthelightmicroscope. Reagent: 1.PBS-T(1000ml) NaCl8gm KH2PO40.2gm Na2HPO41.15gm KCl0.2gm Tween201ml Thimerosol0.1gmImmunostainingofCellsAdherenttoCoverslips
