15 μg of K-562 lysate was run on 6-18% SDS-PAGE under reducing conditions and blotted onto nitrocellulose membrane. antibody at 1 μg/mL was used as the primary antibody and peroxidase conjugated goat anti-mouse IgG was used as the secondary antibody. WT1 band was visualized using ECL Western Blotting Substrate.

Result: antibody can detect WT1 by Western blotting.

IHC-P analysis of testis tissue by WT1 antibody.
IHC-P was performed using sections of the formalin-fixed paraffin-embedded testis tissue. Antigen was retrieved through addition of boiling Tris/EDTA buffer pH 9 in a pressure cooker for 3 min. Endogenous peroxidase activity was quenched by incubating the sections with 3% H₂O₂ for 30 min at room temperature. The sections were then incubated with WT1 antibody at 5 μg/mL at room temperature for 1 h. Poly-peroxidase conjugated goat anti-mouse IgG was used as the secondary antibody. Diaminobenzidine was used as the chromogen. The section was counterstained with hematoxylin. A tissue section incubated with phosphate-buffered saline followed by incubation with the secondary antibody was used as the background control.

Result: Sertoli cell are positively stained at the nuclei.