25 ug total protein per lane of various lysates (see on figure) probed with FOXP3 polyclonal antibody, unconjugated (bs-42173R) at 1:1000 dilution and 4°C overnight incubation. Followed by conjugated secondary antibody incubation at r.t. for 60 min.

4% Paraformaldehyde-fixed MJ (H) cell; Triton X-100 at r.t. for 20 min; Antibody incubation with (FOXP3) polyclonal Antibody, unconjugated (bs-42173R) 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 MJ (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-FOXP3 antibody (bs-42173R): 1 μg/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.