Biotinylated griffonia simplicifolia lectin

Lungs were inflated with 1 mL 1 part 4% PFA/30% sucrose and 2 parts OCT and stored overnight in 4% PFA at 4°C. After 24 hours, lungs were removed from 4% PFA and incubated another 24 hours in 30% sucrose. Lungs were then blocked in OCT and sectioned at 8 µm. H&E-stained lung sections were blindly scored on a 1–5 scale with 5 being the most severe score of pathology using criteria of leukocyte infiltration (1–5) and vascular inflammation/endothelial cell hyperplasia (1–5) for a total score out of 10. Scoring of the lung section was based on the following: 1 – absent, 2 – slight, 3 – moderate (covering up to 5% of total area), 4 – marked (>5% and <10% of total area), and 5 – severe (covering ≥10% of total area). For immunofluorescence staining, sections were incubated with rabbit anti-hResistin (1∶400, generated by Mitchell Lazar), biotinylated Griffonia simplicifolia lectin (1∶400, Vector Laboratories) overnight at 4°C. Sections were incubated with appropriate fluorochrome-conjugated secondary antibodies for 2 hours at 4°C and counterstained with DAPI. Sections were visualized under a Leica microscope (DM5500 B) and Volocity software (PerkinElmer) was used to quantify number of hResistin⁺ and DAPI⁺ cells.