Antigen retrieval solution

The explants at culture day 14 were fixed with phosphate-buffered 4% paraformaldehyde solution for 24 h, and embedded in paraffin. These sections were rehydrated with xylene and graded ethanol solutions. After being treated with 0.3% hydrogen peroxide, the sections were heated at 95 °C for 40 min in antigen retrieval solution (pH 8, Nichirei, Tokyo, Japan). After being treated with skimmed milk solution for 10 min, the sections were treated with mouse anti-HopX antibody (Santa Cruz biotechnologies, Santa Cruz, CA) and rabbit anti-pro-SftpC antibody (Chemicon, Temecula, CA) overnight at 4 °C. After washing, the sections were treated with anti-mouse or anti-rabbit IgG conjugated with the HRP polymer (DAKO, Glostrup, Denmark) for 1 h at room temperature. The sections were further treated with diaminobenzidine-hydrogen peroxide solution, and counterstained with hematoxylin.

To investigate the expression levels of IL-34, M-CSF, CSF1R and CD163 protein in clinical samples from lung cancer patients, tissue sections were stained the in the following manner. TMA slides were immersed in antigen retrieval solution (pH 9.0) (Nichirei, Tokyo, Japan) and boiled for 15 min in an autoclave. Endogenous peroxidase activity was blocked by incubation in 0.3% H₂O₂ in methanol for 15 min. After protein blocking (Catalog No. X0909, Abcam), TMA slides were incubated with a mouse anti-IL-34 antibody (Catalog No. ab101443), a rabbit anti-M-CSF antibody (Catalog No. ab52864, Abcam) in 1:100 dilution, a rabbit anti-CSF1R antibody (Catalog No. HPA012323, SIGMA) in 1:100 dilution or a mouse anti-CD163 antibody in 1:100 dilution (Catalog No. MCA1853, Bio-Rad) in Antibody Diluent (Catalog No. S0809, DakoCytomation) for 30 min at room temperature in a moist chamber. The sections were incubated with HRP-labeled polymer anti-mouse (Catalog No. K4007, DakoCytomation) or anti-rabbit IgG (Catalog No. K4002, DakoCytomation) as the secondary antibody for 30 min at room temperature in a moist chamber. Substrate-chromogen was added, and the specimens were counterstained with hematoxylin.

Paraffin‐embedded tissue sections were deparaffinized, and antigens were retrieved for 5 min in a pressure cooker at 121°C in pH 9.0 antigen retrieval solution (Nichirei Bioscience). For MMP‐12 staining, the sections were incubated with a rabbit monoclonal antibody (1:100 dilution, BS9869M, Bioworld Technology) at room temperature for 60 min. Sites of antibody binding were visualized with the Histofine simple stain MAX‐PO(R) kit (Nichirei Bioscience). 3,3′‐Diaminobenzidine tetrahydrochloride was used as a chromogen, and the sections were counterstained with hematoxylin. For TIMP‐2 staining, a mouse monoclonal antibody (1:100 dilution, 3A4, Santa Cruz Biotechnology) was applied at room temperature for 60 min and visualized with the Histofine mouse staining kit (Nichirei Bioscience). For immunofluorescence, a rabbit polyclonal antibody against p19^ARF (1:300 dilution, ab80, Abcam) and a rat monoclonal antibody against fibroblasts (1:50 dilution, ER‐TR7, Santa Cruz Biotechnology) were used. The sections were visualized with Alexa Fluor 488‐conjugated anti‐rat IgG and Alexa594‐conjugated anti‐rabbit IgG and were counterstained with DAPI.