Sample: Lane 1: Human HeLa cell lysates Lane 2: Human A375 cell lysates Primary: Anti-CD73 (bsm-51239M) at 1/1000 dilution Secondary: IRDye800CW Goat Anti-Mouse IgG at 1/20000 dilution Predicted band size: 63 kDa Observed band size: 63 kDa

Sample: Lane 1: WiDr cell lysates Primary: Anti-CD73 (bsm-51239M) at 1/2000 dilution Secondary: IRDye800CW Goat Anti-Mouse IgG at 1/20000 dilution Predicted band size: 63 kD Observed band size: 72 kD

Sample: Lane 1: MDA-MB-231 cell lysates Primary: Anti-CD73 (bsm-51239M) at 1/4000 dilution Secondary: IRDye800CW Goat Anti-Mouse IgG at 1/20000 dilution Predicted band size: 63 kD Observed band size: 70 kD

Paraformaldehyde-fixed, paraffin embedded (human kidney tissue sections ); 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 (CD73) Monoclonal Antibody, Unconjugated (bsm-51239M) at 1:25 overnight at 4°C, followed by operating according to SP Kit(Mouse)(sp-0024) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (human kidney tissue sections ); 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 (CD73) Monoclonal Antibody, Unconjugated (bsm-51239M) at 1:25 overnight at 4°C, followed by operating according to SP Kit(Mouse)(sp-0024) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (human gastric carcinoma); 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 (CD73) Monoclonal Antibody, Unconjugated (bsm-51239M) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Mouse)(sp-0024) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (Human esophageal 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 (CD73) Monoclonal Antibody, Unconjugated (bsm-51239M) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Mouse)(sp-0024) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (Human kidney); 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 (CD73) Monoclonal Antibody, Unconjugated (bsm-51239M) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Mouse)(sp-0024) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (human tonsil); 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 (CD73) Monoclonal Antibody, Unconjugated (bsm-51239M) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Mouse)(sp-0024) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (human colon carcinoma); 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 (CD73) Monoclonal Antibody, Unconjugated (bsm-51239M) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Mouse)(sp-0024) instructionsand DAB staining.

Blank control:A375. Primary Antibody (green line):Mouse Anti-CD73 antibody (bsm-51239M) Dilution: 1ug/Test; Secondary Antibody (white blue line) : Goat anti-Mouse IgG-AF488 Dilution: 0.5ug/Test. Isotype control（orange line）：Normal Mouse IgG Protocol The cells were 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.