Anti collageniii

Lung paraffin sections were slided into 3μm-thick, dewaxed in xylol 5min for twice, and then rehydrated in 100% ethanol 5min for twice, 75% ethanol 5min and tap water for 5min. The sections were washed by phosphate-buffered saline (PBS) 3min for twice. 3% Hydrogen peroxide and methanol mixed together and the slides were incubated in order to inactive endogenous peroxidases. Then the slides were put into 0.1% pepsin for antigen retrieval. Next procedure was blocked in protein block solution and incubated with primary antibodies: TGF-β1 (Abcam, USA, 1:400), anti-GAP43 (Abcam, USA, 1:1000), anti-TH (Abcam, USA, 1:300), Collagen I (Bioss, China, 1:300), anti-CollagenIII (Bioss, China, 1:300) at 37°C for 2h, then overnight at 4°C. The slides were washed by PBS, then followed by incubation with biotinylated rabbit anti-IgG (ZSGB-BIO, China) and stained with DAB kit (ZSGB-BIO, China) and hematoxylin. The software (Image-pro plus 6.0, Meida Cybernetics LP) was applied to the analysis of the lung section. We measured the density of positive cells in total lung section.

Western blot analysis was performed to detect the expression levels of transforming growth factor-β1 (TGF-β1), collagen I and collagen III; the protocols were as previously described (Wang et al., 2017 (link)). Briefly, 40μg of protein was fractionated by 12% SDS-PAGE and then transferred onto PVDF membranes. The membranes were blocked with 5% non-fat milk for 2 h and then incubated with primary antibodies overnight at 4°C. After being washed, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 2 h. Finally, the membranes were visualized with chemiluminescence reagents (EMD Millipore, Billerica, MA, United States). ImageJ 1.41 Software (NIH, Bethesda, MD, United States) was used to analyze the density of the Western blotting bands. The primary antibodies were as follows: anti-TGF-β1 (1:1000; Abcam, Cambridge, MA, United States), anti-collagen I (1:1000; Abcam, Cambridge, MA, United States) and anti-collagen III (1:1000; Bioss, Beijing, China) antibodies. All protein expression levels were normalized to the GAPDH expression level (1:1000; Goodhere Biotech, Hangzhou, China).