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Tissue/cell: human bladder carcinoma; 4% Paraformaldehyde-fixed and paraffin-embedded; Antigen retrieval: citrate buffer ( 0.01M, pH 6.0 ), Boiling bathing for 15min; Block endogenous peroxidase by 3% Hydrogen peroxide for 30min; Blocking buffer (normal goat serum,C-0005) at 37℃ for 20 min; Incubation: Anti-Phospho-Cyclin B1(Ser147) Polyclonal Antibody, Unconjugated(bs-3122R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining

Blank control (blue line): A549 (blue). Primary Antibody (green line): Rabbit Anti-Phospho-Cyclin B1 (Ser147) antibody(bs-3122R) 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.

Blank control:Hela. Primary Antibody (green line): Rabbit Anti-Phospho-Cyclin B1 (Ser147) antibody (bs-3122R) Dilution: 1μg /10^6 cells; Isotype Control Antibody (orange line): Rabbit IgG . Secondary Antibody : Goat anti-rabbit IgG-AF488 Dilution: 1μg /test. Protocol The cells were fixed with 4% PFA (10min at room temperature)and then permeabilized with 0.1% PBST for 20 min at room temperature. The cells were then incubated in 5%BSA to block non-specific protein-protein interactions for 30 min at room temperature .Cells stained with Primary Antibody for 30 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20,000 events was performed.