Figure 4 Fixation, permeabilization, and staining times are flexible. (A) Jurkat cells were stimulated with PMA (50 nM) and ionomycin (1 mM)(filled histograms) or left untreated (open histograms) and stained for pp38 after fixing with 1.5% formaldehyde for 1, 10, or 30 min and permeabilizing in methanol for 10 min. U937 cells were treated with IFN-γ (50 ng/ml) then fixed as above and stained for pStat1. (B) U937 cells were stimulated with IFN-γ or GM-CSF (10 ng/ml) then fixed with 1.5% formaldehyde at room temperature (20) or 37 for 10 min and permeabilized in methanol for 10 min. Cells were subsequently stained with pStat1 and pStat5 mAbs. (C) Jurkat and U937 cells were left untreated or stimulated with PMA and anisomycin (1 mg/ml) or IFN-g and IL-4, respectively. At the stage of methanol permeabilization, the cells were stored for 10 min, 1 week, or 5 weeks at 20 before staining with the indicated mAbs for 30 min at RT. Unstimulated samples appear as open histograms above stimulated samples that are shown as filled histograms. (D) Jurkat and U937 cells were treated as in part C, then fixed with formaldehyde, permeabilized for 10 min with methanol, and stained for 15, 30, or 60 min at RT with the indicated mAbs. The plots shown are representative of at least two independent experiments.