Af1359

Sections of lung tissues were blocked and then incubated with anti-hResistin (AF1359, R&D Systems), anti-Sirt1 (ab110304, Abcam), anti-F4/80 (ab6640, Abcam), anti-RAGE (ab3611, Abcam), anti-HMGB1 (ab18256, Abcam), or anti-(total) Bruton’s tyrosine kinase (BTK; 8547, Cell Signaling) antibodies, or a combination of two antibodies for double immunofluorescence labeling. Then the sections were incubated with the appropriate fluorochrome-coupled secondary antibodies (Jackson ImmunoResearch) and mounted with ProLong Gold anti-fade reagent with DAPI (P36935, Thermo-Fisher). Staining was imaged and tissue sections were analyzed by confocal microscopy (Leica SPE DMI8). For quantitative analysis, the proportion of area with positive staining was determined with Adobe Photoshop software (Creative Suite 5). Alternatively, positive cells in lung sections from each animal were counted on five randomly chosen high-power fields at 200- or 400-fold magnification. For the study with human tissue samples, quantitative analysis was performed as previously described (33 (link), 34 (link)). Briefly, hResistin-positive cells were counted on 10 randomly chosen visual fields of lung sections in each patient at 400-fold magnification, and the average number of cells in 10 fields was calculated.