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Sample: Lane 1: Mouse Testis tissue lysates Lane 2: Mouse Ovary tissue lysates Lane 3: Mouse Liver tissue lysates Lane 4: Human HL-60 cell lysates Primary: Anti-WT1 (bs-6983R) at 1/1000 dilution Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution Predicted band size: 55 kDa Observed band size: 50 kDa

Sample: TM4 Cell (Mouse) Lysate at 40 ug Primary: Anti-Wilms Tumor Protein (bs-6983R) at 1/300 dilution Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution Predicted band size: 55 kD Observed band size: 48 kD

Tissue/cell: human kidney tissue; 4% Paraformaldehyde-fixed and paraffin-embedded; Antigen retrieval: citrate buffer ( 0.01M, pH 6.0 ), Boiling bathing for 15min; Block endogenous peroxidase by 3% Hydrogen peroxide for 30min; Blocking buffer (normal goat serum,C-0005) at 37℃ for 20 min; Incubation: Anti-WT-1/Wilms Tumor Protein Polyclonal Antibody, Unconjugated(bs-6983R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining

Paraformaldehyde-fixed, paraffin embedded (Mouse 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 (Wilms Tumor Protein) Polyclonal Antibody, Unconjugated (bs-6983R) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (Rat testis); 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 (Wilms Tumor Protein) Polyclonal Antibody, Unconjugated (bs-6983R) at 1:400 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (Mouse testis); 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 (Wilms Tumor Protein) Polyclonal Antibody, Unconjugated (bs-6983R) at 1:400 overnight at 4°C, followed by a conjugated Goat Anti-Rabbit IgG antibody (bs-0295G-FITC) for 90 minutes, and DAPI for nuclei staining.

Blank control:Molt-4. Primary Antibody (green line): Rabbit Anti-Wilms Tumor Protein antibody (bs-6983R) Dilution: 1μg /10^6 cells; Isotype Control Antibody (orange line): Rabbit IgG . Secondary Antibody : Goat anti-rabbit IgG-AF647 Dilution: 1μg /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 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.