Collection of bronchoalveolar lavage fluid (BALF) and histological analysis

Mice were sacrificed 24 h after the last OVA or saline challenge. The lungs were lavaged three times with 0.8 ml of saline, and the collected cells were centrifuged (4°C at 300 × g for 10 min) and subjected to Wright-Giemsa staining as previously reported (27). A number of differential cell counts, including eosinophils, macrophages, lymphocytes and neutrophils, were performed on total of 200 cells based on the staining of characteristics of morphology, as previously described (28). Supernatant samples were collected for cytokine assays. The left lungs were isolated and fixed in 4% paraformaldehyde at room temperature for 24 h, and then embedded in paraffin. Subsequently, 5-µm-thick sections were stained using hematoxylin and eosin (H&E) and observed under a light microscope (magnification, ×200).