Ab212951

Losartan was purchased from Merck Sharp & Dohme Limited (Merck Sharp & Dohme, Australia). AG granule was purchased from Sichuan Baili Pharmaceutical Co. Ltd. (Sichuan, China). NG granule was purchased from China Resources Sanjiu Medical & Pharmaceutical Co. Ltd. (Shenzhen, China). Streptozotocin (STZ) was purchased from Sigma-Aldrich Company (Sigma-Aldrich, USA). Mouse monoclonal anti-Wilms tumor (WT1) (ab212951), rabbit monoclonal anti-Bax (ab32503), rabbit monoclonal anti-Bcl-xl (ab32370), rabbit monoclonal anti-nephrin (ab216341), rabbit monoclonal anti-Nox4 (ab133303), goat anti-rabbit IgG (ab6721), and monoclonal anti-β-actin antibodies (ab8226) were purchased from Abcam Biotechnology (Abcam, England). A rabbit polyclonal anti-α-dystroglycan (abs125549a) antibody was purchased from Absin Biochemical Company (Absin, Shanghai, China). The FastQuant RT Kit (with gDNase) was purchased from Tiangen Biochemical Technology Co. Ltd. (Beijing, China). ChamQ Universal SYBR qPCR Master Mix was purchased from Vazyme Biotech Co. Ltd. (Nanjing, China).

Nephrin and Wilms tumor-1 (WT-1): Following rehydration of deparaffinized sections, antigen retrieval was performed in TRIS–EDTA buffer (pH:9,0) for 20 min in the microwave. After washing with PBS, the sections were incubated in 3% H₂O₂ for 25 min at RT. After washing with PBS, blocking (SensiTek Super Block) was applied for 6 min. Then, the sections were separately incubated with primary antibodies of 1 µg/ml WT-1 (Abcam, ab212951) and 0,607 µg/ml nephrin (Abcam, ab216341) at + 4 ℃ overnight. Following washing with PBS, the instructions of secondary antibody kit (ScyTek Laboratories SensiTek HRP kit) were followed as it was mentioned in staining protocol. Analysis of immunohistochemical stainings was performed on an average 30 glomeruli by evaluating the nephrin staining intensity per glomerulus with the Fiji ImageJ software program and counting the WT-1-positive cells in each glomerulus.