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Paraformaldehyde-fixed, paraffin embedded (Human colon cancer); 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 (BTN2A1) Polyclonal Antibody, Unconjugated (bs-20473R) at 1:400 overnight at 4°C, followed by a conjugated secondary (sp-0023) for 20 minutes and DAB staining.

Blank control:MCF7 cells (blue). Primary Antibody: Rabbit Anti-BTN2A1 antibody(bs-20473R), Dilution: 5μg in 100 μL 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.

Blank control: SH-Sy5Y. Primary Antibody (green line): Rabbit Anti-BTN2A1 antibody (bs-20473R) Dilution: 2μg /10^6 cells; Isotype Control Antibody (orange line): Rabbit IgG . Secondary Antibody : Goat anti-rabbit IgG-AF647 Dilution: 1μg /test. Protocol 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.