Gb11179

Before immunostaining, paraffin-embedded sections were dewaxed and rehydrated in an alcohol series. The sections were washed with phosphate-buffered saline after heat-induced antigen retrieval. Next, the sections were treated with 3% hydrogen peroxide for 15 m to eliminate endogenous peroxidase activity and then incubated with 3% bovine serum albumin for 30 m at 37°C to block non-specific binding sites. Subsequently, sections were incubated overnight at 4°C with diluted primary antibodies, including α-SMA (1:2,000; GB13044; Servicebio, Inc. Wuhan, China), collagen I (1:1,200; GB11022-3; Servicebio, Inc.), matrix metalloproteinase (MMP)-2 (1:1,200; GB11130; Servicebio, Inc.), tissue inhibitors of metalloproteinase-1 (TIMP-1) (1:1,000; 106164-T40; Sino Biological US, Chesterbrook, PA, USA), TGF-β1 (1:500; GB11179; Servicebio, Inc.), and incubated with a conjugated secondary antibody after washing with phosphate-buffered saline (G0002; Servicebio, Inc.). Diaminobenzidine kits (G1211; Servicebio, Inc.) were used to visualize antibody binding areas. For each mouse, we analyzed three liver slices and randomly observed the positive-stained areas in three different fields of vision on each slice. The ratio of positive-stained to total area was calculated by Image-Pro Plus 6.0.

The thorax of mice was fully exposed, and the left lung was washed with PBS and stored in liquid nitrogen for RT-PCR and WB. The right lung was fixed with 4% paraformaldehyde by intratracheal instillation and extratracheal immersion, followed by routine paraffin-embedded sections (4 μm thickness) for pathological observation, including HE staining, Masson staining and immunohistochemistry. HE staining was used to observe the structural integrity of airway wall and alveoli, inflammatory cell infiltration, and smooth muscle hyperplasia in mice to evaluate airway remodeling and emphysema. Alveolar destruction was assessed by calculating MLI as previously described.19 (link) Pulmonary collagen fibril deposition was measured by calculating collagen volume ratio using ImageJ software. Immunohistochemistry was used to assess alveolar secretory function and structural damage, and TGF-β expression.20 (link) Sections were incubated for anti-TGF-β antibody (GB11179, Servicebio), anti-SP-A antibody (ab11579233011, Abcam), anti-SP-D antibody (ab220422, Abcam), anti-ProSP-C antibody (ab211326, Abcam), anti-F4/80 antibody (GB11027, Servicebio). Staining grades were determined by analyzing the number of positive cells and staining intensity using IHC Profiler and scores greater than or equal to 1 were scored as positive. Numbers of macrophage and AECII were counted using IHC ToolBox.