Aluminum potassium sulfate dodecahydrate

Dichloromethane, ethanol, hexane, ethyl acetate, anhydrous sodium acetate, copper (II) sulfate pentahydrate, aluminum potassium sulfate dodecahydrate, calcium carbonate, methanol, aluminum chloride, potassium acetate, Folin-Ciocalteau reagent, sodium carbonate, quercetin, and gallic acid were purchased from Sigma Chemical Co. (Philippines). All the chemicals and reagents were of analytical grade for this study.

Trans-2-[3-(4-tert-Butylphenyl)-2-methyl-2-propenylidene]malononitrile (DCTB), 1,5-diaminonapthalene (DAN), γ-aminobutyric acid (GABA) standard, glutamine standards, Phloxine B, hematoxylin, sodium iodate, and aluminum potassium sulfate dodecahydrate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Corynantheidine standard was extracted from dried Mitragynine speciosa leaves (Pure Land Ethnobotanicals, Madison WI, USA) using methanol or ethanol, and then fractioned and dried using acid-base extraction and column chromatography. Proton nuclear magnetic resonance (¹H NMR), carbon nuclear magnetic resonance (¹³C NMR), high-performance liquid chromatography-photometric diode array (HPLC-PDA), and ultra-performance LC-quadrupole time of flight (UPLC-QTOF) were used to ensure the purity of the standard were 98% or above.^{12 (link)} Epredia^™ Cytoseal^™ mountant XYL, glycerol, Eosin Y intensified solution, μ-opioid receptor polyclonal antibody, donkey anti-rabbit IgG (H+L) highly cross-absorbed secondary antibody (Alexa fluor plus 488, absorption at 497nm, emission at 517nm), and ProLong^™ Gold antifade mountant with DAPI were purchased from ThermoFisher Scientific (Waltham, MA). Xylene was purchased from Honeywell International Inc. (Charlotte, NC).

Mice (9±1 weeks old) were sensitized i.p. on days 0 and 7 with 10 μg ovalbumin (OVA; grade V) adsorbed to 1 mg of aluminum potassium sulfate dodecahydrate (both from Sigma-Aldrich) in 100 μL of saline. They were challenged once a day on days 19-21 in a nebulization chamber with 1% OVA in PBS for 30 min using an Aerotech II jet nebulizer (Biodex) at 10 L/min. Then, they were studied on day 22. A detailed description of the airway mechanics assessment, airway mucin quantification, histology and BALs is provided in the Online Supplementary Methods.