Al oh 3

Female mice (8‐week‐old) were sensitized with an intraperitoneal (i.p.) injection of 10 μg of chicken OVA (Grade V, Sigma‐Aldrich, St. Louis, MO, USA) and 0.3 mg of Al(OH)₃ (SERVA Electrophoresis, Heidelberg, Germany) on Days 1 and 14 and then 12‐week‐old mice were challenged with aerosolized 1% OVA for 30 min on Days 26, 27, and 28. Mice were assessed for airway hyperresponsiveness (AHR) followed by bronchoalveolar lavage (BAL) on Day 30, as previously described (Kibe et al. 2003). Experimental protocols are described in Figure 1.

Germ-free mice were kept on the respective diets for at least two generation. Eight-week-old GF female mice were subcutaneously (s.c.) sensitized on days 1, 14 and 28 with 1 μg of Bet v 1 (Biomay, Vienna, Austria) emulsified in 100 μl of Al(OH)₃ (Serva, Germany). Mice sham treated with Al(OH)₃ alone were used as controls. Seven days after the last immunization, mice were killed by CO₂ asphyxia and samples were taken for further analysis.

Poly-sensitization was done as described previously (28 (link)). Briefly, female BALB/c mice were sensitized with three intraperitoneal injections (days 10, 24, and 39) of a mixture of 5 µg rBet v 1, 5 µg rPhl p 1, and 5 µg rPhl p 5 adsorbed to aluminum hydroxide (Al(OH)₃; Serva, Heidelberg, Germany) (0.68 mg/ml). To induce airway inflammation, one week after the last intraperitoneal immunization, mice were anesthetized by 2% isoflurane in an anesthetic induction chamber and challenged intranasally with 30 µl of a mixture of 5 µg rBet v 1, 5 µg rPhl p 1, and 5 µg rPhl p 5 for 3 consecutive days.
Mice were sacrificed 72 h after the last challenge by exposure to carbon dioxide and blood was collected from the facial vein of mice at the end of the experiment. Sera were collected and stored at −20°C until used.