Interleukin 4 (il 4)

Carboxymethyl cellulose (CMC) sodium salt, DEXA-water soluble, OVA, trypan blue, aluminum hydroxide gel, and Al(OH)₃ were obtained from Sigma-Aldrich (St. Louis, MO, USA). Isoflurane was obtained from Hana Pharm. Co. (Hwaseong, Korea). IL-4, IL-5, and an OVA-specific IgE (OVA-sIgE) ELISA kit were obtained from MyBioSource (San Diego, CA, USA). TRIzol reagent was obtained from Invitrogen (Carlsbad, CA, USA).

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.

Concentrations of TGFβ1, interleukin-4 (IL-4), and prostaglandin E2 (PGE2) in the culture supernatant were determined by using commercially available kits. TGFβ1 (560429; BD Biosciences, San Diego, CA, USA), IL-4 (MBS705670; MyBiosource, San Diego, CA, USA), and PGE2 (514010; Caymen Chemical, Ann Arbor, MI, USA). Primer sequences used for quantitative polymerase chain reaction are as follows: IL-4: forward TGT CCA CGG ACA CAA GTG CGA, reverse TCT CAT GAT CGT CTT TAG CCT TTC C; Cox 2: forward CCA CCC GCA GTA CAG AAA GT, reverse CAG GAT ACA GCT CCA CAG CA; and TGF-β: forward CCC TGG ACA CCA ACT ATT GC, reverse GCA GAA GTT GGC ATG GTA GC. Cycling conditions were 95°C for 5 minutes, 60°C for 25 seconds for 35 cycles.