Renal Tissue Histochemical and Immunohistochemical Analysis

Renal tissues were fixed in 10% neutral buffered formaldehyde or 4% paraformaldehyde, embedded in paraffin and then 4 μm sections were cut and stained with standard periodic acid–Schiff, Masson's trichrome and histological techniques for light microscopic examination[16 (link), 17 (link)]. For immunohistochemical staining, primary antibodies against Anti-TGFBI (1:400, ab170874, Abcam), Anti-TNF alpha (1:400, ab1793, Abcam), Anti-NF-kB p65 (phospho S536) (1:600, ab86299, Abcam), and Anti-oxLDL (1:600, ab14519, Abcam) was used. The samples were then stained with ChemMate^TM Envision+HRP (Envision^TM Detection Kit, Code No: GK500705, Gene Tech, Shanghai) and incubated for 45 min at 37°C. Sections were then stained with diaminobenzidine (DAB) and counterstained with hematoxylin. In six mice per group, more than 20 cortical glomeruli were assessed for each mouse. The positive areas were quantified by Image-Pro Plus version 6.0 software according to methods previously reported [18 (link), 19 (link)].