Overlay histogram showing Jurkat cells stained with ab196186 (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 (ab196186, 1/50 dilution) for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) Alexa Fluor® 647 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 solid-state 25mW red diode laser (635 nm) and 675/30 bandpass filter. This antibody gave a positive signal in Jurkat cells fixed with 4% formaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.
ab196186 staining EEA1 in Jurkat cells. The cells were fixed with 100% methanol (5 min), permeabilised 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 ab196186 at 1/50 dilution (shown in red) overnight at +4°C. Nuclear DNA was labelled in blue with DAPI.Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).