Immunoflourescence of Cultured Cells

Materials

L-lysinecoatedglasscoverslipsorchargedglassslides

NeutralBufferedFormalin(SigmaHT50-128)

0.5%NP40inPBS

(2.5NHClor0.07NNaOHforBrDUstainingonly)

Primaryandsecondaryantibodies

Vectashield(VectorLabs)

Protocol

1)GrowcellsonL-lysinecoatedglassslipsorcytospincellsontochargedglassslides.

2)Fixcellsfor5min.inneutralbufferedformalin.

3)PermeABIlizethenucleusbyincubatingin0.5%NP40inPBSatr.t.

4)Rinsein3changesofPBSforatotalof10minutes.

5)ForBrDUstainingdenaturetheDNAbyoneofthefollowing:

a)soakin2.5NHClat37°Cfor15min,or

b)0.07NNaOHfor2minatroomtemp

5)Add100µLprimaryantibody(titerdeterminedemperically~10xtheconcentrationusedinawestern).Coverwithaglassslipandplaceinahumidifiedchamberatr.t.for1hr.

6)FloatthecoverslipoffbydippingintoajarofPBS,andrinseasin4).

7)Addsecondaryantibodyasin5)and6)above.(e.g.FITC-conjgoatanti-rabbit(1:1000)orBiotinylatedisotypespecificantimouseforimmunoperoxidasestaining).Washasbefore.

8)TominimizequenchingofflourochromemountwithVectashield(Vectorlabs)andcoverwithaglassslip.