2.6. Histological and immunohistochemical analysis for TGF‐β

The kidneys were fixed in 4% formaldehyde at 4°C overnight and then embedded in paraffin. Tissue sections (4 μm) were deparaffinized in xylene and rehydrated in graded alcohol. Masson trichome staining and H&E staining were subjected as previously reported. 25 Tubulointerstitial fibrosis was analysed by the percentage of fibrotic area in tubulointerstitial area using the Image pro plus software (Media Cybernetics, Rockville, MD, USA) in 200X randomly fields.

For immunohistochemical staining of TGF‐β, endogenous peroxidase was blocked with 3% H2O2 and non‐specific proteins were blocked with 10% goat serum or rabbit serum for 30 minutes. The sections were then incubated with indicated primary antibodies at 4°C for overnight, respectively, followed by incubation with an HRP‐conjugated secondary antibody at room temperature for 30 minutes. The sections were further counterstained with haematoxylin and then assessed under a microscope in a blinded manner by two pathologists.