Hela cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum, C-0005) at 37°C for 20 min; Antibody incubation with (Phospho-Cyclin B1 (Ser133)) polyclonal Antibody, Unconjugated (bs-3123R) 1:100, 90 minutes at 37°C; followed by a conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue, C02-04002) was used to stain the cell nuclei.

Blank control (blue line): A549 (blue). Primary Antibody (green line): Rabbit Anti-Phospho-Cyclin B1 (Ser133) antibody (bs-3123R) Dilution: 1μg /10^6 cells; Isotype Control Antibody (orange line): Rabbit IgG . Secondary Antibody (white blue line): F(ab’)2 fragment goat anti-rabbit IgG-FITC Dilution: 1μg /test. Protocol The cells were fixed with 2% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min at room temperature.Cells stained with Primary Antibody for 30 min at room temperature. The cells were then incubated in 1 X PBS/2%BSA/10% goat serum to block non-specific protein-protein interactions followed by the antibody for 15 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20,000 events was performed.