At the end of the experimental period, the ears of 6 mice from each group were removed, fixed in 10% neutral phosphate-buffered formaldehyde, and cut into 3 segments ~2 mm in width. After the ear segments were embedded in paraffin, 2 sections ~3 µm in thickness were obtained from each one. One of the sections was stained with hematoxylin and eosin, while the other one was stained with toluidine blue. Three points on each segment were arbitrarily selected for indicating the apical, central, and basal regions, and the number of inflammatory cells in the subcutaneous tissue of each of the 9 areas (3 ear segments × 3 regions) was counted using a Nikon ECLIPSE 50i light microscope (Nikon Co., Tokyo, Japan). The evaluated area was defined by a perpendicular line extending from the edge of the cartilage of the external ear by a length of 100 µm (for eosinophil count) or 400 µm (for mast cell count) to the epidermal layer. Mast cell degranulation was scored as previously described 17 (link): non-degranulated (0%), mildly degranulated (0-50%), and severely degranulated (>50%).