Pdgfr β

The paraffin section of the kidney was deparaffinized and rehydrated using xylene and alcohol in gradients. Next, the sections were heated using citrate buffer pH 6 for 20 min for antigen retrieval and followed by blocking non-specific antigen using blocking serum for 20 min. Then, the slides were incubated with primary antibodies, PDGFR-β (1:200 dilution, Abclonal, Cat. No. A19531) and α-SMA (1:400 dilution, Sigma, Cat. No. A2547), overnight. On the following day, the slides were incubated with secondary antibody, goat-anti-mouse (Abclonal, Cat, No. AS076), and goat-anti-rabbit (Abclonal, Cat. No. AS039) for 1 h, and DAPI staining for 20 min. Finally, the slides were observed under a confocal microscope (Zeiss, Cat. No. LSM900).

The kidney tissue was embedded in 4-μm thick paraffin sections. Then, it was deparaffinized and rehydrated using xylene and alcohol series. The specimens were stained with SR to measure the fraction area of interstitial fibrosis and with periodic acid-Schiff (PAS) to determine tubular injury. This step was followed by antigen retrieval, blocking peroxidase using 3% H₂O₂ in PBS solution, and blocking non-specific antigen using Background Sniper for immunostaining. The slides were incubated with α-SMA (1:400 dilution, Sigma, Cat. No. A2547) and CD68 (1:400 dilution, Abcam, Cat. No. ab955), PDGFR-β (1:200 dilution, Abclonal, Cat. No. A2180), MCP-1 (1:100 dilution, Abcam, Cat. No. ab25124) as the primary antibodies; TrekAvidin-HRP label, conjugated to anti-rabbit Trekkie universal Link (Biocare Medical^®), as the secondary antibody; and diaminobenzidine tetrahydrochloride (DAB). The α-SMA immunostaining was applied to measure myofibroblast expansion, and CD68 antibody was used for counting macrophage cells. The quantification was performed from 15 fields for each sample at ×400 magnification, using ImageJ software.