We stained 2 μm sections with hematoxylin and eosin (H&E) or rabbit-anti-human intelectin (94–145) antibody (1:400; Phoenix Pharmaceuticals, US) or an isotype matched control rabbit IgG (1:2000; Abmart Shanghai, China) for endobronchial and skin biopsies. For endobronchial biopsies, observers who were blinded to the clinical status of the subjects counted numbers of eosinophils/mm2 submucosa, and calculated basement membrane thickness (BMT). We used the mean of 50 measurements taken over a distance of at least 1 mm to calculate BMT as previously described 57 (link).
Mouse lungs were fixed for 5 min by instillation of 4% paraformaldehyde-PBS (Sigma, USA) through a tracheal catheter at a transpulmonary pressure of 15 cmH2O and lungs were fixed in 4% paraformaldehyde-PBS overnight. Five-μm-thick sections were used for immunohistochemistry with rabbit-anti-human intelectin (94–145) antibody (1:400; Phoenix Pharmaceuticals, USA) or an isotype matched control rabbit IgG (1:2000; Abmart Shanghai, China). Antibodies were detected using the DAB kit (Zhongshan Goldenbridge Biotechnology, China) as directed by the manufacturer. Lung sections were also stained with Periodic acid-Schiff (PAS; Goodbio technology, China) staining for detection of mucus. The number of PAS-staining-positive cells was counted in four random fields for each lung section at ×200 magnification 58 (link).
Mouse lungs were fixed for 5 min by instillation of 4% paraformaldehyde-PBS (Sigma, USA) through a tracheal catheter at a transpulmonary pressure of 15 cmH2O and lungs were fixed in 4% paraformaldehyde-PBS overnight. Five-μm-thick sections were used for immunohistochemistry with rabbit-anti-human intelectin (94–145) antibody (1:400; Phoenix Pharmaceuticals, USA) or an isotype matched control rabbit IgG (1:2000; Abmart Shanghai, China). Antibodies were detected using the DAB kit (Zhongshan Goldenbridge Biotechnology, China) as directed by the manufacturer. Lung sections were also stained with Periodic acid-Schiff (PAS; Goodbio technology, China) staining for detection of mucus. The number of PAS-staining-positive cells was counted in four random fields for each lung section at ×200 magnification 58 (link).