ICC/IF image of ab7291 stained HeLa cells. The cells were 100% methanol fixed (5 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then incubated in 1%BSA / 0.3M glycine in 0.1% PBS-Tween for 1h to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab7291, 1µg/ml) overnight at +4°C. Ab98800, goat anti-mouse IgG, was then added as a secondary bridging antibody, at 1/250 dilution for 1h. Ab150129 Alexa Fluor® 488 donkey anti-goat IgG (H+L) (shown in green) was then used at 1µg/ml for 1h as a tertiary antibody. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

The negative control (inset) is a secondary-only assay to demonstrate low non-specific binding of the secondary antibody.

Overlay histogram showing Jurkat cells stained Goat polyclonal to Ikaros (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal donkey serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (Goat polyclonal to Ikaros, 0.1μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 donkey anti-goat IgG (H&L) (ab150129) at 1/4000 dilution for 30 min at 22°C. Isotype control antibody (black line) was goat IgG (polyclonal) (ab37373, 0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.