Il 13

Blood plasma was collected in EDTA-coated tubes following centrifugation and aliquots were stored for further analysis. The canulated, excised lungs were lavaged three times with 0.5 mL of cold PBS. For fractionation the collected BALF was centrifuged at 1000×g for 10 min at 4 °C and superintend was collected, aliquoted and stored at − 80 °C for further analysis. The remaining cell pellets were re-suspended in 0.2 mL of cold PBS containing 0.1% BSA. From that 20 µL of cell suspension was mixed with equal volume of 0.4% Trypane blue and the total live BAL cells were enumerated by using TC20 automated cell counter (Bio-Rad). Subsequently, 50 µL of cell suspension were cytospun by using Cytospin 2 (Shandon) onto slides for staining procedures. For differential count fixed slides were stained with Camco quik stain II and eosinophils were measured using the BZX700 All-in-one microscopy system (Keyence Inc., Osaka, Japan). Different slide was immunostained for eosinophils (Cat# NBP1-42140; Novus Biologicals, CO) and counter stained with DAPI. Plasma and BALF were analyzed for IgE (Cat# LS-F5589; LSBio, Seattle, WA) and T_H2 cytokines (e.g., IL-13, Cat# MBS355405; MyBio Source, San Diego, CA) levels by specific ELISAs as per the manufacturers’ instructions.

IHC analysis was conducted as previously described [28]. Deparaffinized tissue sections were treated with 3% hydrogen peroxide in methanol for 10 min to remove endogenous peroxidase. Antigen retrieval was carried out with sodium citrate buffer (0.1 M) using the microwave method. The slides were incubated with normal serum to block nonspecific binding and then incubated for 1 h with primary antibodies (diluted 1:100 to 1:200) such as IFN-γ (sc-74104, Santa Cruz Biotechnology, Dallas, TX, USA), IL-12p40 (sc-57258), IL-4 (sc-73318), IL-13 (sc-1776), TNF-α (MyBioSource, San Diego, CA, USA), and IL-6 (Novus Biologicals, Littleton, CO, USA). The slides were incubated for 10 min with biotinylated secondary antibodies (Vector Laboratories, PK-7800, Burlingame, CA, USA) and horseradish peroxidase-conjugated streptavidin. Signals were detected using the 3,3-diaminobenzidine tetrahydrochloride substrate chromogen solution, and the cells were counterstained with Mayer’s hematoxylin.