Sample: Mcf-7 Cell Lysate at 40 ug Primary: Anti- Mesothelin (bs-0300R) at 1/300 dilution Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution Predicted band size: 64 kD Observed band size: 62 kD

Blank control: Hela(blue). Primary Antibody: Rabbit Anti-Mesothelin/AF647 Conjugated antibody (bs-0300R-AF647), Dilution: 0.2μg in 100 μL 1X PBS containing 0.5% BSA; Isotype Control Antibody: Rabbit IgG/AF647(orange) ,used under the same conditions. Protocol The cells were washed twice with phosphate-buffered saline (PBS). The cells were 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-0300R-AF647, 0.2μg /1x10^6 cells) for 30 min on ice. Acquisition of 20,000 events was performed.

Blank control: A549(blue), the cells were fixed with 2% paraformaldehyde (10 min) and then permeabilized with ice-cold 90% methanol for 30 min on ice.. Isotype Control Antibody: Rabbit IgG(orange) ; Secondary Antibody: Goat anti-rabbit IgG-FITC(white blue), Dilution: 1:100 in 1 X PBS containing 0.5% BSA ; Primary Antibody Dilution: 1μl in 100 μL1X PBS containing 0.5% BSA(green).