Optical double headed microscope

Rats were euthanized with chloral hydrate at the end of the experiment and the kidneys were collected for histological sample preparation. Formalin-fixed paraffin-embedded tissue sections were cut and stained with Harris hematoxylin (Sigma-Aldrich, USA) and eosin solution (Sigma-Aldrich, USA). Briefly, slides were deparaffinized in deionized water and then stained with hematoxylin for 3 minutes, rinsed under running tap water, 70 % ethanol and then stained with eosin for 3 minutes before the slides were rinsed and dehydrated in ethanol, cleared in xylene, and then mounted by machine (Dako, Denmark). The slides were histologically evaluated under an optical double-headed microscope (Olympus, Tokyo, Japan).

The paraffin-embedded tissues were cut to 3-μm sections. Dako Envision FLEX+ system (K8012; Dako, Glostrup, Denmark) was used to deparaffinized and epitopes were unmasked in PT link with low pH target retrieval solution (Dako, Denmark). Briefly, the slides were blocked for 5 minutes with peroxidase blocking solution (Dako) at room temperature (RT), and then incubated with rabbit polyclonal Ki67 antibody (cat. no. ab15580; 1:800; Abcam, USA), rabbit polyclonal p53 antibody (cat. no. ab131442; 1:500; Abcam, USA) and rabbit polyclonal sox9 antibody (cat. no. ab26414; 1:1000; Abcam, USA) at 4°C overnight before incubated with rabbit linker (Dako, Denmark) for 15 minutes, horseradish peroxidase (Dako) for 30 minutes at RT. The slides were subsequently stained with 3,3′-diaminobenzidine tetrahydrochloride for 10 minutes, counter-stained with hematoxylin, dehydrated, and mounted in Richard-Allan Scientific Cytoseal XYL (Thermo Fisher Scientific, Waltham, MA, USA) before evaluated under an optical double-headed microscope (Olympus, Tokyo, Japan).