ab194072 staining in HeLa cells. The cells were fixed with 4% formaldehyde (10min), permeabilized in 0.1% Triton X-100 for 5 minutes and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated with ab194072 at a working dilution of 1 in 50 (shown in red) and ab7291 (Mouse monoclonal [DM1A] to alpha Tubulin) at 1µg/ml overnight at +4°C, followed by a further incubation at room temperature for 1h with an Alexa Fluor® 488 Goat anti-Mouse secondary (ab150117) at 2μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
Overlay histogram showing HeLa cells stained with ab194072 (red line). The cells were fixed with 4% formaldehyde (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 (ab194072, 2μl/1x106 cells) for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (Alexa Fluor® 647) (1μl/1x106 cells) for 30 min at 22ºC. Unlabelled sample (blue line) was also used as a control.Acquisition of >5,000 events were collected using a 25mW red solid state diode laser (635nm) and 675/30 bandpass filter.This antibody gave a positive signal in HeLa fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.