4% Paraformaldehyde-fixed A549 (H) cell; Triton X-100 at r.t. for 20 min; Antibody incubation with (NKX2-1) monoclonal Antibody, unconjugated (bsm-42444M) 1:100, 90 min at 37°C; followed by FITC conjugated Goat Anti-Mouse IgG antibody (green, bs-60296G-FITC) 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 A549 (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.), followed by secondary antibody incubation for 40 min at room temperature. Primary Antibody (green):Mouse Anti-NKX2-1 antibody (bsm-42444M,1:100); Isotype Control (orange): Mouse IgG (bs-0296P). Blank control (black): PBS. Acquisition of 20,000 events was performed.