ab185015 staining SQSTM1 in wild-type HAP1 cells, untreated and chloroquine-treated (50μM, 24 hours) and chloroquine-treated SQSTM1 knockout HAP1 cells. 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 overnight at +4°C with ab185015 at 1/500 dilution (shown in green) and ab195889, Mouse monoclonal to alpha Tubulin (Alexa Fluor® 594), at 1/250 dilution (shown in pseudocolor red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab185015 staining SQSTM1/p62 in HeLa cells +/- Chloroquine (50μM, 24 hours). The cells were fixed with 100% methanol (5 min), 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 overnight at +4°C with ab185015 at 1/500 dilution (shown in green) and ab195889, Mouse monoclonal to alpha Tubulin (Alexa Fluor® 594), at 1/250 dilution (shown in pseudocolor red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Overlay histogram showing HeLa cells stained with ab185015 (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 (ab185015, 1/500 dilution) for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) Alexa Fluor^® 488 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 20mW Argon ion laser (488nm) and 525/30 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.

ab185015 staining SQSTM1/p62 in HeLa cells. The cells were fixed with 4% PFA (10min) 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 ab185015 (Alexa Fluor^® 488) at a working dilution of 1 in 100 overnight at +4°C (shown in green). AlexaFluor^® 350 WGA was used at a 1/200 dilution and incubated for 1h with the cells, to label plasma membranes (shown in blue). Nuclear DNA was labelled in red with 1.25 μM DRAQ5™ (ab108410).