Eclipse e600 microscopy system

Heart tissue was fixed in 10% formalin for 24 h, embedded in paraffin, and sectioned at 5 µm. The heart sections were deparaffinized, rehydrated, and then stained with hematoxylin and eosin (H&E).
Cardiac fibrosis was examined by Sirius-red staining for collagen deposition as described previously. [21] (link) The proportion of fibrosis (collagen) was quantitated using a Nikon Eclipse E600 microscopy system as described previously [21] (link).
Standard immunohistochemical and immunofluorescent staining protocols were performed as described previously [22] (link). For immunohistochemical staining, heart tissue sections were stained with p-Nrf2 (1:10,000). After washing, sections were incubated with HRP-conjugated secondary antibody, developed with a DAB (3,3-diaminobenzidine) developing system (Vector Laboratories, Inc., Burlingame, CA), and counterstained with hematoxylin. For immunofluorescent staining, anti-Fyn antibody (1:500, Cell Signaling Technology) was used. The secondary antibodies Cy3-conjugated immunoglobulin G (IgG; at 1:200, Abcam) were applied and counterstained with 4,6-diamidino-2-phenylindole (DAPI, 0.0002% solution, Sigma-Aldrich).

Hematoxylin–eosin, periodic acid–Schiff (PAS), and Masson’s trichrome stainings were performed by conventional histochemical methods described previously [25 (link)]. After the mice were euthanized, full-thickness biopsies were taken and fixed in 4% formaldehyde, and then embedded in paraffin. Tissue slices were subjected to hematoxylin–eosin staining. For PAS staining, the kidneys were harvested and fixed in 10% formalin; 5-μm thick sections were stained with PAS reagent. Immunohistochemistry was performed in paraffin sections and detected in cortical sections with a specific primary antibody and the ABC staining kit, and visualized with the DAB detection kit (both kits from Vector Laboratories Inc., Burlingame, CA, USA). The primary antibody used in the present study was RAS (Sigma; Maixin, Fuzhou, China). After being incubated with the secondary antibody (Proteintech Group, Chicago, IL, USA), 2-μm thick sections were developed with an SP immunohistochemical kit (Maixin, China) to produce a brown product and counterstained with hematoxylin. Histologic evaluation was performed using a Nikon Eclipse E600 microscopy system (Nikon Instruments Inc., Melville, NY, USA) without knowledge of the identity of the various groups. The slides were coded and examined by a pathologist blinded to the study protocol to identify histological alterations.