Histological examination of the lungs
After the methacholine challenge, the lung was resected from mice that were euthanized with high dose pentobarbital (200 mg/kg). Lung tissues were fixed in 10% buffered formalin for 24 h at room temperature and then embedded in paraffin. Serial sections (3-μm-thick) were cut and stained with haematoxylin and eosin (Muto Pure Chemicals Co., Tokyo, Japan).