Rabbit anti wt1

Sections were deparaffinized and rehydrated as described above. For immunostainings of Wilms tumor protein (WT1), cleaved caspase-3 and collagen I, antigen retrieval was performed using Tris/EDTA buffer (pH 9.0, 10 mM; 20 min, 100 °C), citrate buffer (pH 6.0, 10 mM; 20 min, 100 °C) or pepsin (0.4% in 0.1 HCL; 15 min, 37 °C), respectively. Endogenous peroxidase was blocked with 0.12% H₂O₂ in Milli-Q (20 min, RT), followed by incubation with primary antibody diluted in 1% BSA in PBS. Antibodies used were (1) rabbit anti-fibronectin (Sigma-Aldrich, St.Louis, MO); (2) rabbit anti-collagen I (AbD Serotec, Puchheim, Germany); (3) rabbit anti-WT1 (Santa Cruz Biotechnology, Santa Cruz, CA); and (4) rabbit anti-cleaved caspase-3 (Cell Signaling Technology, Danvers, MA). Negative controls for immunohistochemistry included normal sera of the same species as the primary antibody. The immunoreactions were visualized with 3,3-diaminobenzidine (Dako), counterstained with hematoxylin, dehydrated, and mounted. Cell numbers were determined by randomly analyzing 25 glomeruli of each experimental animal. Sections stained for fibronectin and collagen I were digitalized and graded blindly on a scale of I to V depending on the staining intensity: I, none; II, minute; III, moderate; IV, high; V, very high.