Ab139645

Lung tissues were homogenized with a Polytron homogenizer. Protein extracts were lysed by protein lysis buffer and quantified using the Bicinchonic acid protein assay. Samples (20 μg) were separated by Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (8–15%) gel and then transferred to polyvinylidene fluoride membranes. Nonspecific binding proteins were blocked with 5% skim milk for 1 h at RT. The membranes were incubated with primary antibodies against anti-α-SMA (SC-53015; Santa Cruz Biotechnology, TX, USA) and anti-JAM2 (ab139645; Abcam, Cambridge, UK) overnight at 4 °C. The chemiluminescence signal was scanned using the ChemiDOC^TM imaging system (Bio-Rad Laboratories, Hercules, CA, USA).

For immunofluorescence staining, 4-μm-thick lung sections were dewaxed with xylene and rehydrated with a gradient of ethanol. The sections were subjected to antigen retrieval with a citrate buffer bath (pH 6) at boiling temperature and then blocked for endogenous peroxidase activity. After washing with phosphate-buffered saline (PBS), the sections were incubated with primary antibodies against alpha smooth muscle actin (α-SMA) (SC-53015; Santa Cruz Biotechnology, Dallas, TX, USA), JAM2 (ab139645; Abcam, Cambridge, UK), Neutrophil (Neu) (ab2557; Abcam, Cambridge, UK), Macrophage (Mac) (ab56297; Abcam, Cambridge, UK), and dendritic cell (DC) (NB110-85474; Novus Biologicals, Toronto, ON, Canada) overnight at 4 °C. The sections were rinsed with PBS and incubated with secondary FITC- or RFP-labelled antibodies for 30 min. Nuclei were counterstained with 4′,6-diamidino-2-phenylidone (DAPI) and the fluorescence images were captured with a fluorescence microscope (IX-51, Olympus, Japan).