Flow cytometry overlay histogram showing HT1080 cells stained with ab230290 (red line). The cells were fixed with 4 % formaldehyde (10 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10 % normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab230290) (1x10⁶ in 100 µL at 1 µg/ml) for 30 min at 22°C.

The secondary antibody Goat anti-mouse IgG H&L (Alexa Fluor^® 488, pre-adsorbed) (ab150117) was used at 1/2000 for 30 min at 22°C.

Isotype control antibody (black line) was mouse IgG1κ; (ab170190) used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

This antibody gave a positive signal in HT1080 cells fixed with 80% methanol (5 min) / permeabilized with 0.1% PBS-Triton X-100 for 15 min used under the same conditions./

ab30483 stained in A431cells. The cells were fixed with 4% paraformaldehyde (10min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 1 hour at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with ab30483 at 5 µg/ml and ab6046 (Rabbit polyclonal to beta tubulin) at 1 ug/ml overnight at +4°C. The secondary antibodies were ab150117 (colored green) used at 1 ug/ml and ab150087 (pseudo-colored red) used at 2 µg/ml for 1 hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43 µM for 1 hour at room temperature.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, L-Arginine and sodium azide (ab30483).

Overlay histogram showing HT1080 cells stained with ab30483 (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 followed by the antibody (ab30483, 0.1μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was a goat anti-mouse Alexa Fluor® 488 (IgG; H+L) (ab150113) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 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. This antibody gave a positive signal in HT1080 cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, L-Arginine and sodium azide (ab30483).