4% Paraformaldehyde-fixed NIH/3T3 (treated with 200 ng/ml TPA/PMA for 30 minutes) (M) cell; Triton X-100 at r.t. for 20 min; Antibody incubation with (phospho-ERK1/2 (Thr202 + Tyr204)) monoclonal Antibody, unconjugated (bsm-34302R) 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 NIH/3T3 (treated with 200 ng/ml TPA/PMA for 30 minutes) (M) 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-phospho-ERK1/2 (Thr202 + Tyr204) antibody (bsm-34302R;1:100); Secondary Antibody (white blue): Goat anti-Rabbit IgG-BF488 (bs-60295G-BF488): 1 μg/test. Blank control (black): PBS. Acquisition of 20,000 events was performed.