Six to eight week old female BALC/c mice, obtained from SLAC, were immunized twice, at weekly intervals, with 0.4 ml saline containing 100 μg OVA (Ovalbumin, Sigma-Aldrich, St Louis, MO, USA) mixed with 0.2 ml Freund’s Complete Adjuvant (F5881-10 ML; Sigma-Aldrich). For subsequent injections, the Ag dose was reduced to 50 μg of OVA per injection. One week after the second immunization at day 14, an intranasal challenge was performed under light ether by applying 50 μl of OVA in saline solution (10 μg) or saline alone as a control [18 (link)]. rHa24-GST, GST (negative control) and Mepyramine (340 μg, m.w. 402; Sigma-Aldrich; positive control), was administered intratracheally with 50 μl saline buffer under ketamine anesthesia, 1 h before the OVA challenge. BAL (Bronchoalveolar lavage) was performed 3 days after the intranasal challenge by cannulating the trachea under ketamine anesthesia and washing two times with 1 ml ice-cold PBS. The BAL was centrifuged, and the supernatant was frozen for histamine. The cell pellet was re-suspended in PBS and counted by a hemocytometer.
After BAL, the mice were sacrificed (3 days after OVA challenged). The whole lung was removed and fixed in 4 % paraformaldehyde for Hematoxylin & Eosin and Periodic acid Schiff reagent staining.
After BAL, the mice were sacrificed (3 days after OVA challenged). The whole lung was removed and fixed in 4 % paraformaldehyde for Hematoxylin & Eosin and Periodic acid Schiff reagent staining.