thick sections were stained with Giemsa, Periodic acid-Schiff or Azan Mallory. Sections of lung tissue were stained immunohistochemically to detect ��-smooth muscle actin , platelet endothelial cell adhesion molecule-1 /CD31. Mouse monoclonal anti-smooth muscle , or ready-to-use mouse monoclonal CD31 was used as a primary antibody. For immunohistochemical staining, the VECTASTAIN ABC Kit and the Vector M.O.M. Immunodetection Kit were used according to the manufacturers�� protocols. Diaminobenzidine was used as a substrate for the immunoperoxidase reaction. Sections were lightly purchase 24276-84-4 counterstained with hematoxylin and analyzed by bright-field microscopy. An OLYMPUS BX61 microscope with Scion Image software was used for the morphological analysis. Eosinophils were counted according to a previously described method . The thickness of subepithelial fibrosis and smooth muscle layer were analyzed as follows. The area of subepithelial fibrosis and smooth muscle layer around a bronchus was measured. The average thickness was determined by the area of the positive layer divided by the length of the internal circumference of the area. The mean values for thickness were calculated for 8�C10 bronchi per left lung lobe. To quantify the microvessel density, the number of CD31-positive blood vessels per field was counted in 5 random fields in each sample. To analyze the effects of GSK-1278863 IMD-4690 on subepithelial fibrosis, we histologically examined subepithelial fibrosis and collagen deposition. In Azan-Mallory-stained sections, subepithelial fibrosis in Dp/CMC mice was increased compared with control/CMC mice . Subepithelial thickness was significantly decreased by IMD-4690 administration in Dp/IMD mice . Similarly, the increased total collagen in the lungs of Dp/CMC mice was reduced by IMD-4690 treatment . In the Giemsa-stained sections, the number of infiltrating eosinophils in the subepithelium increased in Dp/CMC mice compared to control/CMC mice . Treatment with IMD- 4690 significantly decreased the numbers of infiltrating eosinophils . The Periodic acid-Schiff staining also showed mucous metaplasia in airway epithelial cells in Dp/ CMC mice, and the number of PAS-positive cells
