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.

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.

ab30483 staining human breast cancer cells by ICC/IF.  Cells were PFA fixed and blocked with 1% serum for 16 hours at 20°C prior to incubating with ab30483 (at 30µg/ml) for 16 hours at 4°C.  An Alexa Fluor® 488 conjugated goat anti-mouse antibody, diluted 1/200, was used as the secondary.

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