2.6. Histological Analysis of Lung Tissue in OVA or AST Exposed Mice

The left lungs of the mice were removed, transferred into 4% formalin for 24 h (room temperature) and subsequently transferred into PBS. The left lung was dehydrated using ethanol and xylene, embedded in paraffin and 4 μm sections were obtained. The paraffin embedded lung sections were stained with hematoxylin and eosin (H&E), periodic acid-schiff (PAS), and pico sirius red. Images of the lung tissue sections stained with H&E and PAS were acquired with a microscope equipped with a ×20 or ×40 objective lens. For immunohistochemistry, the paraffin-embedded sections were deparaffinized. The slides were washed at room temperature and hydrated. The endogenous peroxidase activity was then quenched with 3% hydrogen peroxidase. The sections were then blocked and the endogenous avidin and biotin were blocked, following the manufacturer’s instructions. The samples were then stained with an antibody against caspase-1 or caspase-3. Biotinylated secondary antibodies (2 μg/mL) were used and were detected with horseradish peroxidase, using a Vectastain Elite ABC (Vector Laboratories). Inflammatory cells/epithelium, PAS positive cells, lung fibrosis and the expression of caspase were analyzed by the Image J program [18].