Kidney tissues were fixed in 10% formalin for 24 h, embedded in paraffin, and sectioned at 5 μm thickness for pathological examination and immunofluorescent staining. The sections were deparaffinized and rehydrated, stained with hematoxylin and eosin (H&E). Periodic acid-Schiff (PAS) staining was used to examine the glycogen content of renal tissues, as described previously [25 (link)]. Renal fibrosis was evaluated by Sirius-red and Masson’s staining for collagen [22 ,26 (link)]. Briefly, sections were stained with mixture of 0.1% Sirius Red F3BA and 0.25% Fast Green FCF. We used the Sigma-Aldrich Trichrome Staining Kit for Masson’s staining.
Standard immunofluorescent staining protocols were performed according to previous studies [27 (link)]. Anti-Nrf2 antibody (1:400 dilution, Santa Cruz Biotechnology, Dallas, TA, USA) and anti-Fyn antibody (1:400 dilution, Cell Signaling Technology, Boston, MA, USA) were used, and the secondary antibodies Cy3-conjugated immunoglobulin G (IgG; 1:200 dilution, Abcam, Cambridge, MA, USA) were applied for 1 h and counterstained with 4,6-diamidino-2-phenylindole (DAPI; 1:2000 dilution, Sigma-Aldrich) for 10 min at the room temperature. The slices were covered with aqueous mounting medium (Sigma-Aldrich) and analyzed under a fluorescence microscope (Nikon, Tokyo, Japan). All sections were evaluated using a Nikon Eclipse E600 microscopy system.
Standard immunofluorescent staining protocols were performed according to previous studies [27 (link)]. Anti-Nrf2 antibody (1:400 dilution, Santa Cruz Biotechnology, Dallas, TA, USA) and anti-Fyn antibody (1:400 dilution, Cell Signaling Technology, Boston, MA, USA) were used, and the secondary antibodies Cy3-conjugated immunoglobulin G (IgG; 1:200 dilution, Abcam, Cambridge, MA, USA) were applied for 1 h and counterstained with 4,6-diamidino-2-phenylindole (DAPI; 1:2000 dilution, Sigma-Aldrich) for 10 min at the room temperature. The slices were covered with aqueous mounting medium (Sigma-Aldrich) and analyzed under a fluorescence microscope (Nikon, Tokyo, Japan). All sections were evaluated using a Nikon Eclipse E600 microscopy system.