The kidneys of rats were removed, fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5 μm sections. The sections were deparaffinized, hydrated, and stained with hematoxylin and eosin (H&E), Periodic Acid-Schiff (PAS), and Masson Trichrome, respectively. Renal injury and morphological disorders were observed under a microscope (Olympus, Tokyo, Japan). Semiquantitative scoring was performed to assess extent and intensity of extracellular matrix deposition and fibrosis in the glomeruli and tubulointerstitium using an arbitrary unit: 0, normal; 1, mild; 2, moderate; and 3, severe [22 (link)].
A TUNEL assay was carried out using the In Situ Cell Death Detection Kit (Roche, Shanghai, China) according to the manufacturer's instructions. Briefly, renal tissue sections were treated with 0.1% Triton X-100 (Servicebio, Wuhan, China) for 20 min at room temperature, blocked with 3% H2O2, and then placed in working solution (10% enzyme solution and 90% label solution) for 1 hour at 37°C. The apoptotic cells were stained dark brown by adding diaminobenzidine (DAB; Servicebio, Wuhan, China) and observed under a microscope (Olympus, Tokyo, Japan).
A TUNEL assay was carried out using the In Situ Cell Death Detection Kit (Roche, Shanghai, China) according to the manufacturer's instructions. Briefly, renal tissue sections were treated with 0.1% Triton X-100 (Servicebio, Wuhan, China) for 20 min at room temperature, blocked with 3% H2O2, and then placed in working solution (10% enzyme solution and 90% label solution) for 1 hour at 37°C. The apoptotic cells were stained dark brown by adding diaminobenzidine (DAB; Servicebio, Wuhan, China) and observed under a microscope (Olympus, Tokyo, Japan).
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