ab30394 staining Integrin beta 1 in wild-type HAP1 cells (top panel) and Integrin beta 1 knockout HAP1 cells (bottom panel).

The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab30394 at 10μg/ml concentration and ab202272 at 1/250 dilution (shown in pseudo colour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Mouse IgG (Alexa Fluor^® 488) (ab150117) at 2 μg/ml (shown in green). Nuclear DNA was labeled in blue with DAPI.

This data was developed using the same antibody clone in a different formulation containing PBS and azide (ab30394).

Overlay histogram showing HAP1 wildtype (green line) and HAP1-ITGB1 knockout cells (red line) stained with ab30394.

Live HAP1 wildtype and HAP1-ITGB1 knockout cells were incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab30394, 1µg/0.5x10⁶ cells) for 30 min at 22°C. A mouse IgG1 isotype control antibody (ab170190) was used at the same concentration and conditions as the primary antibody (HAP1 wildtype - black line, HAP1-ITGB1 knockout - grey line). Unlabeled sample was also used as a control (this line is not shown for the purpose of simplicity). Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter.

This data was developed using the same antibody clone in a different formulation containing PBS and azide (ab30394).

ab30394 stained HeLa (Human epithelial cell line from cervix adenocarcinoma) cells.

The cells were fixed in 4% formaldehyde for 10 minutes at room temperature and then incubated in 1% BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1hour at room temperature to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab30394 at 10µg/ml) overnight at +4°C. The secondary antibody (pseudo-colored green) was a Goat Anti-Mouse Alexa Fluor^® 488 (IgG H&L) preadsorbed (ab150117) used at a 1/1000 dilution for 1hour at room temperature. Alexa Fluor^® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1hour at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.

This data was developed using the same antibody clone in a different formulation containing PBS and azide (ab30394).

Overlay histogram showing HeLa (Human epithelial cell line from cervix adenocarcinoma) cells stained with ab30394 (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 goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab30394, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was a goat anti-mouse DyLight^® 488 (IgG; H+L) ab96879 at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 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 4% paraformaldehyde (10 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 formulation containing PBS and azide (ab30394).