STAININGNONFILAMENTOUSACTINWITHVITAMIN-DBINDINGPROTEIN

Materials

1.Gc-globulin(vitamin-Dbindingprotein;Calbiochem345802),prepare2mg/mlstockinPBSsolutionA,clarify25,000rpmfor20minina42.2Tirotor.

2.PBS/BSA:PBSsolutionAwith1%BSA(BoehringerMannheim100350)and0.1%NaN₃,storedat4^oC.

3.DilutedGc-globulin,dilute2mg/mlstockwithPBS/BSAtoobtain1-10ug/ml.Need200ulpercoverslip.

4.PrimaryantibodiesagainstGc-globulin(DAKOA021),diluted1:500withPBS/BSA.

5.Secondarygoatanti-rabbitantibodies,typicallydiluted1:100.

Procedure

1.Fixandextractcellsfollowingtheformaldehyde-acetoneprotocol.

2.Washthecoverslipfor10mininPBS/BSA.

3.CutapieceofParafilmtomatchtheareaofstainingandpipetdilutedGc-globulinonthepiece.Invertthecoverslipontotheparafilm.Preparea100mmplasticpetridishcontainingapieceofwetfilterpaper.Place2woodensticksinthedishandputcoverslipupsidedownonthesticks.Letsitatroomtemperaturefor60min.

4.Washgently3x,10mineachwithPBS/BSAonashaker.FillacoverslipboxwithPBS/BSAandsinkthecoversliptothebottom.Thecoveringparafilmshouldfloatoff.

5.StainwithantibodiesagainstGc-globulinandsecondaryantibodies(seeImmunofluorescenceprotocol).

Materials

1.PBS/BSA:PBSsolutionAwith1%BSA(BoehringerMannheim100350)and0.1%NaN₃,storedat4^oC.

2.Fluorescein-Gc-globulin.Dilutedto10ug/mlinPBS/BSA.

Procedure

1.Fixandextractcellsfollowingtheformaldehyde-acetoneprotocol.

2.Washthecoverslipfor10mininPBS/BSA.

3.Cutapieceofparafilmtomatchtheareaofstainingandpipetdilutedfluorescein-Gc-globulinonthepiece.Invertthecoverslipontotheparafilm.Preparea100mmplasticpetridishcontainingapieceofwetfilterpaper.Place2woodensticksinthedishandputcoverslipupsidedownonthesticks.Letsitatroomtemperaturefor60min.

4.Washgently3x,10mineachwithPBS/BSAonashaker.FillacoverslipboxwithPBS/BSAandsinkthecoversliptothebottom.Thecoveringparafilmshouldfloatoff.

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