Fluorochrome coupled secondary antibodies

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.

Immunofluorescence staining was carried out as described previously (37 (link)). Briefly, the paraffin sections were blocked with appropriate blocking serum (Vector Laboratories, Burlingame, CA) for 1 hour at room temperature and then treated with anti-FIZZ1 (Abcam, Cambridge, UK) and anti-SMA (Dako) antibodies. Then the sections were incubated with the appropriate fluorochrome-coupled secondary antibodies (Jackson ImmunoResearch, West Grove, PA). Finally, the sections were washed in PBS, mounted with ProLong® Gold antifade reagent with DAPI (Invitrogen, Grand Island, NY), and covered and sealed with a glass coverslip. Negative control sections for the immunohistochemical experiments received identical treatments but were not exposed to the primary antibody; they showed no specific staining. Staining was imaged with a Zeiss 510 Meta confocal microscope (Carl Zeiss Microscopy, Thornwood, NY) at the Johns Hopkins School of Medicine Microscope Facility.