Overlay histogram showing HeLa cells stained with ab53358 (red line). The cells were fixed with 4% paraformaldehyde (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. The cells were then incubated with the antibody (ab53358, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight^® 488 goat anti-rat IgG (H+L) (ab98386) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rat IgG2a, kappa monoclonal [aRTK2758] (ab18450, 2µg/1x10⁶ cells ) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HeLa cells fixed with 80% methanol (5 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.