Nbp1 42140

Tissue sections, 3–4 μm in thickness, were deparaffinized in CitriSolve (Decon Labs., King of Prussia, PA) and rehydrated by processing them through graded alcohol solutions. Antigen unmasking was accomplished by digesting tissue in 10 μg/ml of protein kinase for 15 min at room temperature (Fisher Scientific, Waltham, MA). Endogenous enzymes and non-specific background were blocked with Background Punisher (Biocare Medical, Pacheco, CA), followed by BLOXALL (Vector Laboratories, Burlingame, CA). The primary antibody (NBP1–42140; Novus Biologicals, Centennial, CO) was incubated on the tissue sections at a dilution of 1:100 for 1 h at room temperature. Subsequently, sections were sequentially incubated with an alkaline phosphatase-based detection polymer kit (MACH 4; Biocare Medical), and Warp Red (Biocare Medical). Sections were counterstained with Tacha’s hematoxylin (Biocare Medical) and mounted using a permanent mounting medium (EcoMount; Biocare Medical).

The formalin-fixed lungs were then processed for paraffin-embedding and lung tissue sections were prepared. Histochemical staining with hematoxylin and eosin (H&E) was carried out to assess the gross lung morphology. Serial sections were also stained with Alcian Blue (Richard-Allan Scientific) and Periodic acid Schiff’s reagent stain (AB/PAS) to stain for mucin glycoproteins as described earlier^{17 (link)}. The airway epithelial cell and mucous cell numbers per mm basal lamina^{5 (link)} were measured using the BZX700 All-in-one microscopy system (Keyence Inc., Osaka, Japan). Separate sections were immunostained for airway mucin MUC5AC (Cat# MAB2011; Millipore Inc., MA) and eosinophil (Cat# NBP1-42140; Novus Biologicals, CO) following dual-staining technique as described earlier^{18 (link)}. In all cases, quantification and morphometry was carried out by a person unaware of the slide identity.