α smooth muscle actin α sma

Lung sections (5 μm) were deparaffinized, rehydrated through a graded alcohol series, and exposed to a microwave-based antigen retrieval with a citrate buffer (10 mM of sodium citrate, pH 6.0 for 15 min). Endogenous peroxidases were quenched using 3% H₂O₂ for 5 min. The sections were incubated with surfactant protein-B (SPB; 1:200) or α smooth muscle actin (α-SMA; 1:200) (both from Boster Biological Technology, Wuhan, China) antibodies at 37°C for 2 h. The primary antibody was omitted in the negative control samples. After washing with PBS, the sections were incubated with poly-peroxidase-conjugated anti-mouse/rabbit IgG for 30 min at 37°C using the Polymer-HRP Detection System (Zymed Laboratories, South San Francisco, CA, USA) according to the manufacturer’s instructions. The slides were visualized with diaminobenzidine (DAB; Dako, Glostrup, Denmark), counterstained with Mayer’s hematoxylin, dehydrated through increasing concentrations of alcohol, cleared in xylene, and mounted in neutral balsam (Sigma).

When the density of CFs reached 70–80%, the supernatant was removed and the cells were washed three times with PBS. The cells were fixed with 4% paraformaldehyde at room temperature for 20 min and permeabilized with 0.1% Triton X-100 for 15 min. The cells were blocked with 5% bovine serum albumin (BSA) for 30 min at 37°C and incubated with primary antibodies against α-smooth muscle actin (α-SMA) (1 : 200; Boster, Birmingham, USA), vimentin (1 : 100; Cell Signaling Technology, Danvers, USA), collagen I (1 : 100; Boster), periostin (1 : 100; Proteintech, Rosemont, USA), CD31 (1 : 50; Arigo, Taiwan), and Angptl4 (1 : 100; Cell Signaling Technology) overnight at 4°C. The next day, the fluorescence-conjugated secondary antibodies (1 : 1,000; Cell Signaling Technology) were incubated with cells for 1 h at 37°C. The nuclei were counterstained with Hoechst 33342 (Sigma-Aldrich, Saint Louis, USA), and the images were viewed under a fluorescence microscope (OLYMPUS DP73, Tokyo, Japan).