bcl-2-PE staining kit: no saponin in ready-to-use solution

From: Karim Vermaelen (Karim.Vermaelen@rug.ac.be)
Date: Fri Aug 11 2000 - 04:54:20 EST 

Hi everybody,
Small technical question:
I'm using the Pharmingen bcl-2-PE staining kit for the detection of
murine bcl-2. Cells have to be fixed and permeabilized of course. Next,
the infosheet suggests "20 µl of MAb solution per million cells".
However, the buffer in which the ready-made MAb is diluted contains no
permeabilizing agent. Could this have an impact on the penetration of
the antibody? (I got very poor signal). How fast does the cell membrane
"close" again after witdrawal of perm-buffer? Is it safe to override the
method on the infosheet and dilute the MAb in a saponin-buffer? How far
should these ready-made MAb solutions be diluted? It'd be great to hear
from anybody with experience in such staining protocols.

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