25 ug total protein per lane of various lysates (see on figure) probed with Transferrin monoclonal antibody, unconjugated (bsm-33243M) at 1:5000 dilution and 4°C overnight incubation. Followed by conjugated secondary antibody incubation at r.t. for 60 min.

Paraformaldehyde-fixed, paraffin embedded (human skin 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 (Transferrin) Monoclonal Antibody, Unconjugated (bsm-33243M) at 1:400 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 (Transferrin) Monoclonal Antibody, Unconjugated (bsm-33243M) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Mouse)(sp-0024) instructionsand DAB staining.

Blank control（black line）:HepG2. Primary Antibody (green line): Mosue Anti-Transferrin(Serum Loading Control) antibody (bsm-33243M) 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 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.