Thisprotocolsallowstheuseoffluorescencemicroscopytovisualizeepitope-taggedreceptorsexpressedbystableadherentcelllines.IncontrasttothepermeABIlizedcellprotocol,thisprotocolwhereantibodyis"fed"toliveunpermeabalizedcellswillonlyvisualizereceptorsthatstartedoutonthesurfaceofthecell.

Materials:

WashSolution--TotalVolume2liters;ManiatusTBSwith1mMCaCl2,16gNaCl,6gTrisbase,0.4gKCl,0.294gCaCl2.2H2O;pH7.4withconc.HCl.

Blotto--TotalVolume10mL,0.3gdrymilk(3%),100ul10%TritonX-100(0.1%),100ul100mMCaCl2(1mM),500ul1MTris-ClpH7.5(50mM).

Fixative--approximately2mLpercoverslip37%formaldehydediluted1:10withPBS(150mMNaCl,10mMNaPi,pH7.4)

Procedure:

1.Usinga6welltissuecultureplate,add2mLofmediatoeachwelltobeused.

2.Equilibratemediainincubator(~30-45").

3.Addcoverslips.Equilibrateinincubator(~25").

4.Addprimaryantibodyat1:500to1:1000dilutiontomediafor30".Exampleantibodies:M1mouseanti-Flagantibody(Sigma,Covance)(Ca+2sensitive)1:350-1:500;12CA5mouseanti-HA(Roche)1:1000polyclonal

5.Treatcells.Typicalincubationisapproximately25".

6.Quicklyaspiratemediaandaddfixative(~2mL/well).Fixfor15-20".

7.Wash3x.

8.Aspiratethefinalwash,takingcaretodrytheedgesofthecoverslipwell.AddBlottotoonlythecoverslip(~50uL)andincubate20".VARIATION:SomepeopleprefertransferringthecoversliptoasheetofParafilminatissueculturedishwithasmallamountofwetwhatmaninside.Theparafilmhelpstheantibodysolution"beadup"onthecoverslip,andthewetwhatmanpaperhelpspreventtheantibodyfromdryingup.

9.AspirateBlotto,andapplysecondaryAb.Incubate20"inthedark.DiluteantibodywithBlotto.Cy3donkeyanti-mouse(JacksonImmunoresearch)1:500,FITCgoatanti-mouse1:500,TexasRedgoatanti-mouse1:500.

10.Wash3x.

11.Wetmountwithanti-fadesolution,i.e.VectaShield(VectorLabs)orCitifluor(TedPella).Putsmalldropofmountingmediaonglassslide.Useforcepstopickupcoverslipandaspirateanyexcessliquid.Placecoverslipcell-sidedownontoglassslides.Aspirateanyexcessmountingmediafromedgesofcoverslip.Sealedgeswithnailpolish.

References:

JeffreyL.Benovic(Editor).RegulationofGProtein-CoupledReceptorFunctionandExpression(ReceptorBiochemistryandMethodology).

EpitopeTagging,BasicLaboratoryMethods.PDFmanualfromRochewebsite.