Anti collagen type 3

Kidney specimens, stored at -80° C, were thawed at room temperature, and kidney cryosections were performed according to standard procedures. NRK-52E cells grown on coverslips underwent 4% formalin fixation for 1h. Upon blocking with 5% bovine serum albumin (BSA) at ambient, anti-E-cadherin (14472; Cell Signaling Technology, USA), anti-α-SMA (55135; Proteintech, China) and anti-collagen type III (22734; Proteintech) primary antibodies were added for incubation at 4° C overnight. This was followed by staining with FITC– or Cy3–linked secondary antibodies (Proteintech). DAPI (4′,6-diamidino-2-phenylindole) counterstaining was carried out, and samples were assessed under a Leica DM4000B fluorescence microscope (Leica, Germany).

The content of TNF-α, collagen type I, and collagen type III in infarcted myocardium was assessed by Western blot. The protein concentrations were measured with BCA protein Assay Kit (Beyotime Biotechnology, China). Equal amounts of protein (20 μg) were added into each lane of a 10% SDS-PAGE (New Cell & Molecular Biotech, China) and separated. Then, the protein was transferred onto PVDF membranes (Millipore, United States). And the membranes were blocked by Protein Free Rapid Blocking Buffer (EpiZyme, China) for two h and incubated with primary antibodies (anti- TNF-α, 1:1000, Abclonal, China; anti-collagen type I, 1:1000, Proteintech, United States; anti-collagen type III, 1:5000, Proteintech, United States; anti-GAPDH, 1:1000, Abclonal, China) at 4 ℃ for 12 h. After incubation with HRP-coupled secondary antibody (goat anti-rabbit or rabbit anti-mouse IgG, 1:10,000, Boster, China), the membranes were treated with chemiluminescence (Solarbio, China) for 3 min and visualized on a Visionwork system.