Wilms tumor protein 1 wt1

Kidney sections were boiled in citric acid buffer (10 mM, pH 6.0) for 20 min for antigen retrieval. The sections were blocked in 10% normal horse serum and incubated with a primary antibody (1:100 diluted) against Wilms’ Tumor Protein 1 (WT1) from Boster Biological Technology (Pleasanton, CA, USA) overnight at 4 °C. The sections were incubated with biotinylated secondary antibody (Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature. The sections were incubated in avidin-biotin-peroxidase complex solution (ABC solution; Vector Laboratories) for 30 min and then developed using a 3,3′-diaminobenzidine (DAB) Peroxidase Substrate Kit (Vector Laboratories) for 30 s. The sections counterstained with hematoxylin were analyzed by using a CKX41 light microscope (Olympus). The number of stained nuclei was counted from 10 images of 200× microscopic fields per kidney section from each group.

The 10% formalin-fixed and paraffin-embedded kidney tissue sections (5 μm-thick) were prepared. Briefly, the fixed kidney sections were deparaffinized, rehydrated and antigen-retrieved in sodium citrate buffer (10 mM, pH 6.0) for 20 min. The sections were blocked in 10% normal horse serum and incubated with a primary antibody against CD68 (Cluster of Differentiation 68) from (Abcam) or Wilms’ Tumor Protein 1 (WT1) from Boster Biological Technology (Pleasanton, CA, USA) overnight at 4 °C. The sections were incubated with a biotinylated secondary antibody (Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature. The sections were incubated in avidin-biotin-peroxidase complex solution (ABC solution, Vector Laboratories) for 30 min and developed using 3,3′-diaminobenzidine (DAB) Peroxidase Substrate Kit (Vector Laboratories). Then, the sections were counterstained with Mayer’s hematoxylin and analyzed using a CKX41 light microscope (Olympus). The number of CD68-stained cells or WT1-stained nuclei (equivalent to the number of podocytes) was counted from 10 images of 400× magnification per kidney section from each group, (n = 3), using ImageJ software (NIH).