Overlay histogram showing HeLa cells untreated (red line) and HeLa cells treated with Chloroquine, 50µM, 24 hours, (green line) stained with ab225454. The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab225454, 1/5000 dilution) for 30 min at 22°C.

Isotype control antibody (black line) was Rabbit IgG (monoclonal) Phycoerythrin (ab209478) 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 50 mW Yellow/Green laser (561nm) and 586/15 bandpass filter.

This antibody gave a positive signal in HeLa +/- Chloroquine cells fixed with 4% formaldehyde (10 min)/permeabilized with 0.1% PBS-Triton X-100 for 15 min used under the same conditions.