Overlay histogram showing HT1080 cells stained with ab25406 (red line). The cells were fixed with 4% paraformaldehydel (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab25406, 0.5µg/1x106 cells) for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 (1µg/1x106 cells ). Acquisition of >5,000 events was performed. This antibody gave a positive signal in HT1080 cells fixed with 80% methano/permeabilized in 0.1% PBS-Tween used under the same conditions.