Aluminum potassium sulfate

The protocol applied to establish OVA-induced allergic airway disease in mice was as described in a previous study [24 (link)]. Briefly, mice were randomly distributed into four groups (n = 6 each): control (Con), OVA, rGal-1, and Dex. The mice were sensitized by intraperitoneal injection of 10 μg OVA (Chicken Egg OVA, Grade V; Sigma) and 1 mg aluminum potassium sulfate (Sangon Biotech, Shanghai, China) in 0.5 ml saline on day 0, then challenged for 30 min per day with 1% aerosolized OVA on days 14–17. Control mice were saline-sensitized and challenged with nebulized saline solution. On days 14–17, rGal-1 and Dex mice were pretreated with recombinant Gal-1 protein (3 μg/animal, Peprotech EC Ltd., London, UK) or dexamethasone (1 mg/kg, Sigma-Aldrich) diluted in 0.1 ml of sterile saline 30 min prior the OVA challenge. The doses of rGal-1 and Dex were described previously [11 (link), 13 (link)]. All of the mice were killed 24 hours after the final challenge. The left lung was harvested and subjected to bronchoalveolar lavage and subsequent differential cell counting and ELISA analysis, and the right lung was used for histopathological analysis and further Western blotting assays.

The mice were euthanized by cervical dislocation. The mammary glands were dissected, spread onto a glass slide, fixed in methanol (10%), dehydrated in graded solutions of ethanol, cleared in xylene overnight, hydrated in graded solutions of ethanol, and stained in carmine alum staining solution [0.2% carmine (Sigma) and 0.5% aluminum potassium sulfate (Sangon Biotech)] for about 2 days until the stain has infiltrated the tissues. Finally, these were dehydrated in graded solutions of ethanol, cleared in xylene overnight, and mounted with a coverslip. The slides were scanned, and digital pictures were taken by Perkin Elmer Vectra Slide Analysis System which was powered by inForm^® software (BS, MT, United States).

The murine model of allergic airway inflammation was established according to previous study (27 (link)). Briefly, the mice (6–8 weeks old) were sensitized on day 0 with an intra-peritoneal injection of OVA (Sigma, St. Louis, MO, USA) emulsified with 1 mg potassium aluminum sulfate (Sangon Biotech, Shanghai, China) in 500 μl of saline. Airway inflammation was induced by inhalation of 1% aerosolized OVA 30 min per day, for consecutive 7 days. Control mice received saline-sensitization and inhalation of nebulized saline solution. The mice from WT-OVA-Melatonin group were administered with Melatonin (10 mg/kg, Sigma), and the mice from TLR2^−/−-OVA-Luzindole group were administered with Luzindole (30 mg/kg, Sigma) 1 h before allergen-challenge through intra-peritoneal injection, respectively, the dose of Melatonin and Luzindole was used according to previous studies (19 (link), 28 (link)). Experiments were performed with six mice per group. Mice were harvested 24 h following the last challenge.