Hi Bill, I assume that you mean that you cant use acid etc to unwind the DNA as that will destroy the GFP fluorescence? The way I get round that is to use DNase to unwind the DNA which is a much milder way of doing it. The basic protocol to start with is found in Carayon and Bord JIM (1992), 147, 225-30. The fixation is paraformaldehyde/Tween. I have found that the fixation can be quite critical - make sure that the pf concentration isnt too low and that the pH is right. After fixation in 1% pf/0.01% Tween, wash, add 50kUnitz DNase in 1ml of Ca/Mg containing PBS, incubate 30' at 37¡C, wash, primary Ab, wash, 2¡ Ab and analyse. I usually use BDs BrdU ab and then come in with a Cy3 second layer (I get a better separation than using PE). Once you get the thing working it is a good technique and especially useful in GFP cells. Good luck! Derek On Tue, 27 Jul 1999, Bill Throndset wrote: > I am looking for BrdU incoporation protocols other than Becton Dickinson's > protocol. I have tried twice with B.D.'s protocol but it didn't work for my > Hela cells. Because my Hela cells express GFP, I can't use direct labeling. > I used Pharmigen's anti-BrdU, Biotin-mIgG1 as secodary antibody, and SA-APC > or SA-perCP. ************************************************************************ Derek Davies Voice: (44) 0171 269 3394 FACS Laboratory, FAX: (44) 0171 269 3100 Imperial Cancer Research Fund, e_mail: derek.davies@icrf.icnet.uk London, UK Web Page: http://www.icnet.uk/axp/facs/davies/index.html In tenebris lux *************************************************************************
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