Sample: placenta(Mouse) Lysate at 40 ug Primary: Anti-VNN1(bs-10314R) at 1/300 dilution Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution Predicted band size: 52 kD Observed band size: 52 kD

Tissue/cell: rat spleen tissue; 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-VNN1 Polyclonal Antibody, Unconjugated(bs-10314R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining

Blank control: 293T(blue). Primary Antibody:Rabbit Anti-VNN1 antibody(bs-10314R), Dilution: 5μg in 100 1μL 1X PBS containing 0.5% BSA; Isotype Control Antibody: Rabbit IgG(orange) ,used under the same conditions ); Secondary Antibody: Goat anti-rabbit IgG-PE(white blue), Dilution: 1:200 in 1 X PBS containing 0.5% BSA. Protocol The cells were washed twice with phosphate-buffered saline (PBS).The cells were then incubated in 1 X PBS containing 0.5% BSA + 1 0% goat serum (15 min) to block non-specific protein-protein interactions followed by the antibody (bs-10314R, 5μg /1x10^6 cells) for 30 min on ice. The secondary antibody used was Goat Anti-rabbit IgG/PE antibody at 1/200 dilution for 30 min on ice. Acquisition of 20,000 events was performed.

Blank control: 293T. Primary Antibody (green line): Rabbit Anti-VNN1 antibody (bs-10314R) Dilution: 1μg /10^6 cells; Isotype Control Antibody (orange line): Rabbit IgG . Secondary Antibody : Goat anti-rabbit IgG-PE Dilution: 1μg /test. Protocol The cells were incubated in 5%BSA to block non-specific protein-protein interactions for 30 min at 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.

Blank control: A431. Primary Antibody (green line): Rabbit Anti-VNN1 antibody (bs-10314R) Dilution: 2μg /10^6 cells; Isotype Control Antibody (orange line): Rabbit IgG . Secondary Antibody : Goat anti-rabbit IgG-PE Dilution: 1μg /test. Protocol The cells were fixed with 4% PFA (10min at room temperature)and then permeabilized with 90% ice-cold methanol for 20 min at-20℃. The cells were then incubated in 5%BSA to block non-specific protein-protein interactions for 30 min at 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.