Hematoxylin and eosin stain, Alcian blue stain, and quantification.

Staining was performed on paraffin-embedded sections and analyzed as described in previous studies (40). Briefly, for general histological analysis, lung sections were stained with hematoxylin and eosin (H & E) and histological grading based on infiltration of white blood cells and perivascular thickness. Separate sections were stained with Alcian blue (to stain mucin) and fast nuclear red (to identify cell nuclei). Quantification of inflammation and mucin staining was performed by histological grading, as previously described (40). For histological grading of inflammation, a score of 1 to 4 was given, with 1 being no inflammation present, 2 being one ring of leukocytes around the vasculature and mild infiltration, 3 being two to three rings and moderate infiltration, and 4 being four or more rings and numerous leukocytes throughout the epithelial tissue. Epithelial thickness was quantified using National Institutes of Health (NIH) ImageJ to measure the distance from the basolateral surface of the epithelial cell to the muscular layer. Mucin-positive cells were quantified by calculating the percentage of total epithelial cells per millimeter that stained positive with Alcian blue. Because Alcian blue can also stain basal lamina matrix proteins, staining at the basal lamina was not considered positive. Mucin staining was calculated using a user-defined NIH ImageJ macro; images were inverted, and total red channel density (corresponding to blue by eye) per 100 cells was determined.