Figure 2 Differences in fixation and permeabilization techniques suggest an optimal protocol. (A) Ten fixation/permeabilization methods (perm methods) were tested for their ability to show maximal shift in phospho-epitope staining in response to stimulation of the model cell lines. The FACS histograms show one representative experiment in which U937 cells were treated with IFN-g (50 ng/ml) for 10 min, then fixed with 1.5% formaldehyde (PFA) (methods 4-10), permeabilized with various reagents for 10 min, and stained with pStat1 Ax488 for 30 min. Open histograms represent unstimulated cells; filled plots indicate treated cells. Note there were large differences between organic solvents (methods 4-6) and typical permeabilization reagents such as saponin and Triton X-100 (methods 7-10) for pStat1 staining. (B) Summary of fold changes measured with phospho-mAbs by the ten permeabilization methods. U937 cells were stimulated with IFN-g (50 ng/ml) and GMCSF (10 ng/ml) for 10 min, and stained with pStat1 Ax488 and pStat5 Ax647. Jurkat cells were exposed to PMA (50 nM) and ionomycin (1 mM) for 10 min and analyzed for pERK1/2 (Ax488) and pp38 (Ax647) levels. Data represent the average of three independent experiments, in which the trends shown were extremely consistent. The right axis represents fold change of pStat1. Fold change = MFIstim/ MFIunstim.