Figure 5 Phospho-epitopes are largely stable both before and after staining. (A) Jurkat cells were treated with PMA or anisomycin (filled plots) or left unstimulated (open plots). They were fixed and permeabilized with methanol, then stained immediately with pERK Ax488 or pp38 PE, or incubated in staining medium (PBS + 1% BSA) overnight at 4, before subsequent staining. (B) U937 cells were stimulated with IFN-g or GM-CSF (filled plots) then fixed, permeabilized, and stained with pStat1 Ax488 and pStat5 Ax647. The cells were incubated in staining medium at 4 for 4-16h before FACS analysis.