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Sample: U937 Cell (Human) Lysate at 30 ug Primary: Anti- Cyclin B1 (bs-0572R) at 1/300 dilution Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution Predicted band size: 48 kD Observed band size: 50 kD

Sample: U251 Cell (Human) Lysate at 30 ug Primary: Anti- Cyclin B1 (bs-0572R) at 1/300 dilution Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution Predicted band size: 48 kD Observed band size: 50 kD

Sample: Lane 1: Lymph node (Rat) Lysate at 40 ug Lane 2: Testis (Rat) Lysate at 40 ug Lane 3: Spleen (Rat) Lysate at 40 ug Primary: Anti-Cyclin B1 (bs-0572R) at 1/1000 dilution Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution Predicted band size: 55-60 kD Observed band size: 60 kD

Paraformaldehyde-fixed, paraffin embedded (Mouse small intestine); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (Cyclin B1) Polyclonal Antibody, Unconjugated (bs-0572R) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Tissue/cell: human colon 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-Cyclin B1 Polyclonal Antibody, Unconjugated(bs-0572R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining

Paraformaldehyde-fixed, paraffin embedded (Rat esophageal); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (Cyclin B1) Polyclonal Antibody, Unconjugated (bs-0572R) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

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 (Cyclin B1) polyclonal Antibody, Unconjugated (bs-0572R) 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-Cyclin B1 antibody（bs-0572R）. 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.

Cell: Hela Concentration:1:100 Host/Isotype:Rabbit/IgG Flow cytometric analysis of primary antibody (Cat#: bs-0572R) on Hela(green) compared with isotype control in the absence of primary antibody (blue) followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG(H+L) secondary antibody .