2.4. Histology and immunohistochemistry assay

Four‐μm thick sections from paraffin‐embedded mouse kidney were stained with haematoxylin and eosin (HE), Sirius Red Stain and Masson's trichrome Staining (MTS) to evaluate kidney structure and fibrosis injuries based on the manufacturers’ instructions. Tubular injury score was graded from 0 to 4 based on the cortico‐medullary region percentage as previously described 29 : 0, no damage; 1, <25%; 2, 25%‐50%; 3, 50%‐75%; and 4, >75%. The fibrotic positive area of the murine kidney was determined by calculating the percentage of colour‐pixel count in the entire field containing the cortico‐medullary region. The renal injury and fibrotic area were evaluated by two pathologists and five randomly selected fields of each slide were quantified at 200× magnification. Histology and immunohistochemistry (IHC) staining was performed according to the manufacture's protocols as previously described. Briefly, each slide from the sample was deparaffinized and the antigen was retrieved. After incubating with antibodies (Table S1) overnight at 4°C, each tissue was incubated with horseradish peroxidase‐conjugated secondary antibody and stained with 3,3‐diaminobenzidine tetrahydrochloride (DAB, Maixin, China). The positive area of each slide was photographed under a microscope (Olympus, Japan) at 200× magnification in five random fields, and the results were analysed by Image Pro Plus 6.0.