Anti α sma

The proteins of cells or lung tissues were lysed in radioimmunoprecipitation assay buffer lysis buffer with phenylmethanesulfonyl fluoride (Servicebio, Beijing, China). Protein concentrations were quantified using BCA protein quantification kit (Beyotime, Shanghai, China). Protein samples were segregated using sodium dodecyl sulfate polyacrylamide gel electrophoresis, then transferred to 0.45-μm polyvinylidene difluoride membranes. Membranes were blocked in tris-buffered saline and 0.1% Tween 20 with 5% bovine serum albumin at room temperature for 2 hours. The membranes were incubated with primary antibodies at 4°C overnight. The primary antibodies were used: anti-SPARC (1:200; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-GAPDH (1:5,000; Abcam, Cambridge, MA, USA), anti-E-cadherin (1:1,000; Cell Signaling Technology, Danvers, MA, USA), anti-N-cadherin (1:1,000; Cell Signaling Technology), anti-fibronectin (1:1,000; HUABIO, Hangzhou, China), anti-p-Smad2 (1:1,000; HUABIO), anti-Smad2 (1:1,000; HUABIO), anti-collagen I (1:1,000; Abcam), anti-collagen III (1:1,000; Abcam), anti-α-SMA (1:1,000; HUABIO). The second day, the bands were incubated with goat anti-mouse or goat anti-rabbit IgG (secondary antibodies) for 1 hour at room temperature. Then the bands were visualized to chemiluminescence by using enhanced chemiluminescent reagent.

The lung tissues were fixed in 4% paraformaldehyde 48 hours. After dehydration, tissues were embedded in paraffin, and 5-μm-thick paraffin sections were cut as slides for pathological staining like hematoxylin and eosin (H&E), Periodic Acid Schiff (PAS), and Masson’s trichrome.
Immunohistochemical staining was performed using standard procedures. After deparaffinization and hydration using xylene and sequentially decreasing concentrations of ethanol, the sections were repaired in antigen repair solution and treated with 3% hydrogen peroxide (H₂O₂) to remove endogenous peroxides. Then, the sections were blocked with goat serum for 1 hour. The samples were incubated with primary antibodies (anti-SPARC [1:200; HUABIO], anti-E-cadherin [1:200; Cell Signaling Technology], anti-N-cadherin [1:200; Cell Signaling Technology], anti-fibronectin [1:200; HUABIO], anti-collagen I [1:200; BOSTER], anti-collagen III [1:200; Abcam], anti-α-SMA [1:200; HUABIO]) overnight at 4°C, followed by HRP-labelled secondary antibody at 37°C for 30 minutes. Color development was performed with 3,3-diaminobenzidine solution, and the reaction was stopped with PBS.