Evaluating Mucus and MMP-9 in Lung Tissues

Lung tissues were processed as described previously (17 (link)). To evaluate mucus production and airway inflammation, lung tissue was stained with periodic acid-Schiff (PAS; IMEB Inc. San Marcos, CA) or hematoxylin and eosin (H&E; BBC Biochemical, Mount Vemon, WA). The score of mucus production was calculated as the percentage of purple (positive) stained cells in the total cell count. MMP-9 protein was stained using an immunohistochemistry (IHC) kit (Abcam) following the manufacturer’s instructions. A primary antibody against MMP-9 (1:300; Cell Signaling Technology, Danvers, MA) was used for IHC staining. Brown stained cells are positive for MMP-9. Quantitative analyses were conducted with a light microscope in a blinded manner (Leica Microsystems) at 10× and 20× objective lenses using the IMT i-Solution software (Vancouver, North Road Burnaby, Canada).