Sample: Hela（human）Cell Lysate at 40 ug MDA-MB-231（human） Cell Lysate at 40 ug Primary: Anti-CK7 (bsm-33060M) at 1/2000 dilution Secondary: IRDye800CW Goat Anti-Mouse IgG at 1/20000 dilution Predicted band size: 54 kD Observed band size: 54 kD

Sample: Lane 1: Human HeLa cell lysates Lane 2: Human HepG2 cell lysates Lane 3: Human A549 cell lysates Lane 4: Human HUVEC cell lysates Primary: Anti-CK7 (bsm-33060M) at 1/1000 dilution Secondary: IRDye800CW Goat Anti-Mouse IgG at 1/20000 dilution Predicted band size: 54 kDa Observed band size: 60 kDa

Paraformaldehyde-fixed, paraffin embedded (human breast); 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 (CK7) Monoclonal Antibody, Unconjugated (bsm-33060M) 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 esophagus); 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 (CK7) Monoclonal Antibody, Unconjugated (bsm-33060M) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Mouse)(sp-0024) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (rat colon); 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 (CK7) Monoclonal Antibody, Unconjugated (bsm-33060M) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Mouse)(sp-0024) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (rat bladder); 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 (CK7) Monoclonal Antibody, Unconjugated (bsm-33060M) 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 uterus); 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 (CK7) Monoclonal Antibody, Unconjugated (bsm-33060M) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Mouse)(sp-0024) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (Mouse bladder); 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 (CK7) Monoclonal Antibody, Unconjugated (bsm-33060M) 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); 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 (CK7) Monoclonal Antibody, Unconjugated (bsm-33060M) 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 lung 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 (CK7) Monoclonal Antibody, Unconjugated (bsm-33060M) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Mouse)(sp-0024) instructionsand DAB staining.

HepG2 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 (CK7) monoclonal Antibody, Unconjugated (bsm-33060M) 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:Hela. Primary Antibody (green line): Mouse Anti-CK7 antibody (bsm-33060M) Dilution: 1ug/Test; Secondary Antibody : Goat anti-mouse IgG-FITC Dilution: 0.5ug/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 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:Hela. Primary Antibody (green line): Mouse Anti-CK7 antibody (bsm-33060M) Dilution: 1ug/Test; Secondary Antibody : Goat anti-mouse IgG-FITC Dilution: 0.5ug/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 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.