Hep-G2 (H) cells were treated with or without CoCl2 (500uM) for 6 h, 25 μg total protein per lane of cell lysates (see on figure) probed with HIF-1 Alpha monoclonal antibody, unconjugated (bsm-62518R) at 1:1000 dilution and 4°C overnight incubation. Followed by conjugated secondary antibody incubation at r.t. for 60 min.

4% Paraformaldehyde-fixed HepG2 (H) （HepG2 treated with 500uM CoCl2 for 6 hours）cell; Triton X-100 at r.t. for 20 min; Antibody incubation with (HIF-1 Alpha) monoclonal Antibody, unconjugated (bsm-62518R) 1:100, 90 min at 37°C; followed by conjugated Goat Anti-Rabbit IgG antibody (green, bs-60295G-BF488) at 37°C for 90 min, DAPI (blue, C02-04002) was used to stain the cell nuclei. PBS instead of the primary antibody was used as the blank control.

4% Paraformaldehyde-fixed Hela (H) （HeLa treated with 500uM CoCl2 for 6 hours）cell; Triton X-100 at r.t. for 20 min; Antibody incubation with (HIF-1 Alpha) monoclonal Antibody, unconjugated (bsm-62518R) 1:100, 90 min at 37°C; followed by conjugated Goat Anti-Rabbit IgG antibody (green, bs-60295G-BF488) at 37°C for 90 min, DAPI (blue, C02-04002) was used to stain the cell nuclei. PBS instead of the primary antibody was used as the blank control.

The Hela( treated with 500uM CoCl2 for 6 hours) (H) cells were fixed with 4% PFA (10 min at r.t.) and then permeabilized with 90% ice-cold methanol for 20 min at -20℃,the cells then were incubated in 5%BSA to block non-specific protein-protein interactions (30 min at r.t.).Primary Antibody (green):Rabbit Anti-HIF-1 Alpha antibody (bsm-62518R): 1:50-100/10^6 cells; Secondary Antibody (white blue): Goat anti-Rabbit IgG-BF488 (bs-60295G-BF488): 1 μg/test. Isotype Control (orange): Rabbit IgG (bs-0295P). Blank control (black): PBS. Acquisition of 20,000 events was performed.