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ZK03186小鼠αL岩藻糖苷酶(AFU)ELISA试剂盒_
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FLUORESCENTSTAININGOFCELLS

Materials

1.Fluorescentphalloidininmethanol.Phallacidindoesnotworkaswell.Dilute10ul330nMstockinto500ulPBSforeachlargecoverslip.

2.PBS,solutionA.

Procedure

1.FixandpermeABIlizecells(seeotherprotocols).Mountcoverslipontoaplasticframereservedforfixedsamples.

2.Turnofflight.Dilutefluorescentphalloidin50xintoPBS.

3.Gentlypipetphalloidinsolutionontocoverslip.Stainfor30minatroomtemperature.

4.Rinsecoverslip3xwithPBS.

5.FillthechamberwithPBSoranantIBLeachingsolutionandobserve.Dishesmaybestoredat4oCinasealed,light-tightcontainer.

Materials

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.

Procedure(donotallowcoverslipstodryoutanytime)

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.

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