Mip 2

The BALF extraction supernatants were analyzed for Il-6, TNF-α, MCP-1 (eBioscience), IL-1β, and MIP-2 (Abcam) concentration by an enzyme-linked immune sorbent assay (ELISA) kit following the manufacturer’s instructions.16 (link)

Paraffin-embedded tissue sections (4-µm thick) were fixed with 4% paraformaldehyde on slides for 2 h at 37°C. The sections were washed with PBS three times and then stained with the appropriate antibody: SEMA-7A (1:1,000; cat no. ab23578; Abcam) for 12 h at 4°C. Samples were incubated at 4°C for 12 h, washed with PBS, and then incubated with a specific fluorescence-conjugated IgG (1:1,000; cat no. O-6382, Invitrogen; Thermo Fisher Scientific, Inc.) for 1 h in a light-protected chamber at 37°C. Subsequently, sections were counterstained with Von Willebrand factor (2 µg/ml, Sigma-Aldrich; Merck KGaA) or DAPI (2 µg/ml, Sigma-Aldrich; Merck KGaA) at 37°C for 2 h and mounted. In addition, tissue sections (4-µm thick) were incubated with IL-1β (1:1,000; cat no. ab200478), IL-17A (1:1,000; cat no. ab79056), TNF-α (1:1,000; cat no. ab6671), CXCR2 (1:1,000; cat no. ab14935), MIP-2 (1:1,000; cat no. ab9950), KC (1:1,000; cat no. ab798362; all Abcam) at 4°C for 12 h. Immunofluorescence signals were detected using a laser scanning confocal microscope (Carl Zeiss AG, Oberkochen, Germany).