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琼脂糖胶原复合水凝胶的制备及性能研究
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1.Gc-globulin(vitamin-Dbindingprotein;Calbiochem345802),prepare2mg/mlstockinPBSsolutionA,clarify25,000rpmfor20minina42.2Tirotor. 2.PBS/BSA:PBSsolutionAwith1%BSA(BoehringerMannheim100350)and0.1%NaN3,storedat4oC. 3.DilutedGc-globulin,dilute2mg/mlstockwithPBS/BSAtoobtain1-10ug/ml.Need200ulpercoverslip. 4.PrimaryantibodiesagainstGc-globulin(DAKOA021),diluted1:500withPBS/BSA. 5.Secondarygoatanti-rabbitantibodies,typicallydiluted1:100. 1.Fixandextractcellsfollowingtheformaldehyde-acetoneprotocol. 2.Washthecoverslipfor10mininPBS/BSA. 3.CutapieceofParafilmtomatchtheareaofstainingandpipetdilutedGc-globulinonthepiece.Invertthecoverslipontotheparafilm.Preparea100mmplasticpetridishcontainingapieceofwetfilterpaper.Place2woodensticksinthedishandputcoverslipupsidedownonthesticks.Letsitatroomtemperaturefor60min. 4.Washgently3x,10mineachwithPBS/BSAonashaker.FillacoverslipboxwithPBS/BSAandsinkthecoversliptothebottom.Thecoveringparafilmshouldfloatoff. 5.StainwithantibodiesagainstGc-globulinandsecondaryantibodies(seeImmunofluorescenceprotocol). 1.PBS/BSA:PBSsolutionAwith1%BSA(BoehringerMannheim100350)and0.1%NaN3,storedat4oC. 2.Fluorescein-Gc-globulin.Dilutedto10ug/mlinPBS/BSA. 1.Fixandextractcellsfollowingtheformaldehyde-acetoneprotocol. 2.Washthecoverslipfor10mininPBS/BSA. 3.Cutapieceofparafilmtomatchtheareaofstainingandpipetdilutedfluorescein-Gc-globulinonthepiece.Invertthecoverslipontotheparafilm.Preparea100mmplasticpetridishcontainingapieceofwetfilterpaper.Place2woodensticksinthedishandputcoverslipupsidedownonthesticks.Letsitatroomtemperaturefor60min. 4.Washgently3x,10mineachwithPBS/BSAonashaker.FillacoverslipboxwithPBS/BSAandsinkthecoversliptothebottom.Thecoveringparafilmshouldfloatoff.STAININGNONFILAMENTOUSACTINWITHVITAMIN-DBINDINGPROTEIN
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