Tgf β1

The 3 μm-thick renal tissue sections were incubated with citrate antigen retrieval solution for 20 min at 95°C. Thereafter, the sections were stained with a monoclonal antibody against transforming growth factor-β1 (TGF-β1; Cat# GB13028; dilution 1 : 500; Servicebio, Wuhan, China) and α-smooth muscle actin (α-SMA; Cat# GB111364; dilution 1 : 500; Servicebio). The sections were incubated with goat antirabbit antibodies overnight and then with a secondary antibody for 50 min. The positive areas were visualized using a DAB Kit (Servicebio). The integrated optical density of protein expression was calculated using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Total ovarian proteins were extracted by using RIPA lysis buffer containing protease inhibitors and protein phosphatase inhibitors (Servicebio Biotechnology Co., Ltd., Wuhan, China). The mixtures were homogenized and stored on ice for 10min, and then centrifuged at 12,000 rpm for 10 min at 4° C. The supernatant was used to conduct western blot analysis. The proteins (30 μg) were separated with 10% SDS-polyacrylamide gels and then transferred onto PVDF membranes (Millipore, Burlington, USA). After being blocked with 5% skimmed milk for 1 h, the strips of target proteins and internal protein were washed with TBST. The strips were each incubated with primary antibodies against TGF-β1, p-Smad3, Smad3, Smad7, MMP2, and GAPDH purchased from Servicebio Biotechnology Company (Wuhan, China) overnight at 4° C. The strips were then incubated with HRP-conjugated anti-rabbit IgG (CST, Danvers, USA) secondary antibodies for 2h at room temperature. The contents of target proteins were detected with an enhanced chemiluminescence agent (Tanon, Shanghai, China). The grey values of the protein strips were analyzed with Image J software (National Institutes of Health, Bethesda, USA). The expressions of target proteins were normalized to the protein of GAPDH.