immunocytometry of active bax(6A7) - (Jun/13/2009 )

dear all
I am currently performing immunocytometry of active bax(6A7). I have tried a fixation in 2% formaldehyde followed by permeabilization with digitonin (100 microg/ml) on ice for 10 min. then staining with Bax Ab (in BSA/PBS) for 90 min on ice. but the peak of positvely stained cells was not clearly separated from that of backgound flourescence.
doesanyone use a better protocol? and is there a method to check the optimal fixation and permeabilization conditions without using the antibody?
thanks

How long is your fixation?
" " is your staining?
I usually do:

-Induce apoptosis
-Fix cells 30' with 4% PF at RT
-Rinse twice with cold PBS
-Incubate with digitonin (same concentration as you)
-Rinse twice with cold PBS
-Incubate OVERNIGHT with 1st Ab at 1:500 at 4ºC in blocking buffer (BSA 3%)
-Rinse 3x with cold PBS
-Incubate with 2nd Ab at 1:2000 in blocking buffer + SAPONIN (is very important because effect of digitonin is not for ever, you need to add again a detergent if not the cell is not permeable anymore).
-Rinse with PBS and resuspend on blocking buffer for FACS analysis.

Hope it works!!

I haves tested 2 cell lines with my protocol. one of them gave bad results but the other gave good results. bad results mean both the signals of Bax Ab and positive control Ab overlayed each other suggesting a non specific binding of the Ab in that cell line. The point is now that what is an aletrnative strategy to overcome such non specific binding?

what do you mean with your positive control antibody?
I don't really get it. Some things could be:
-overexpress Bax-GFP and perform the treatment and check oligomerization under microscope.
-gated cells are correct? don't forget apoptotic that change in forward and side scatter characteristics.
I need more info to help you... sorry!

sorry for the mistake , I ment isotype control