When I performed intracellular staining (Ki-67,DAKO) I used the following protocol (bcl-2,DAKO did not work well): Staining of intracellular antigens for FACS-analysis The surface antigens (extracellular) of 2 x 10 5 cells were stainde as usually (e.g. CD19/CD3). After the last washing step the cells were resuspenden in 400 µl PBS, 0,5% BSA. To permeabilize the cells icecold 100% ethanol was added drop by drop (vortexing!). The suspension was then incubated on ice, in the dark for at least 30 minutes. The cells wete then washed with 2 ml PBS, 0,5%BSA and resuspended in 100 µl of " cell cycle solution" [Anderson et al., 1995]. For intracellular steining the respectiv antibodies was added.(positive contro : stainingwith Ki-67) Cell cycle solution: 01% Tween 20 0,1 mMol EDTA in PBS Christa Amrehn Institut fuer Pathologie Uni Wuerzburg Josef-Schneider Str. 2 97080 Wuerzburg 49/0931/201-3790 path132@mail.uni-wuerzburg.de
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