Avidin peroxidase complex

Quantification of IL-4, IL-5, and IL-13 in the BALF was accomplished by using a commercially available ELISA kit, according to the manufacturer’s instructions (eBioscience, San Diego, CA, USA). For immunoglobulin (Ig) analysis, the serum was harvested 24 h after the final OVA challenge to determine the levels of OVA-specific IgE and IgG1 by ELISA. Briefly, 96-well plates (Corning Costar, New York, NY, USA) were coated with 200 μg/mL of OVA diluted in 0.1 M NaHCO₃ (pH 8.3). After 3 h of incubation at 37°C, the plates were washed 6 times and blocked with 10% FBS in PBS for 1 h at 37°C. Serum samples were then incubated overnight at 4°C. After 6 washes with PBS containing 0.05% Tween-20, biotinylated antibodies against mouse IgE or IgG1 (Biolegend, San Diego, CA, USA) were added to the respective wells. After incubation for 1 h at room temperature, the plates were washed 6 times and incubated with an avidin-peroxidase complex (Sigma) for 30 min at room temperature. After the final washes, tetramethylbenzidine (Sigma) was added and the color was allowed to develop for 15–20 min. The reaction was stopped by adding 2 M H₂SO₄ and the absorbance intensity was read at 450 nm using a microplate reader (Tecan, Clontarf, Australia). Serum levels of OVA-specific Igs are expressed as the optical density.

For the analysis of TSLP expression in tissue slices, 2–4 µm thick sections were used without further pre-treatment (anti-mouse/rat TSLP; anti-Iba1), or after antigen retrieval for 1 hour in a commercial food steamer in 10 mM EDTA buffer pH 8.5 (anti-GFAP). The sections were rinsed in 0.05 M Tris-buffered saline (TBS) and incubated in Dako buffer (DakoCytomation, Vienna, Austria) containing 10% fetal calf serum (DB/FCS) for 20 min. The slides were incubated in blocking buffer at 4°C overnight with the following primary antibodies: polyclonal rabbit anti-mouse/rat TSLP (ab3, Sigma Aldrich; 1:50), polyclonal rabbit anti-Iba1 (Wako Chemicals GmbH, Neuss, Germany; 1:50), or mouse anti-rat glial fibrillary acidic protein (GFAP, Neomarkers, Fremont, CA; 1:200). The sections were then washed three times in TBS and incubated with biotinylated secondary antibodies (donkey anti-rabbit, 1:2,000, or sheep anti-mouse, 1:500; both antibodies from Jackson Immunoresearch) in DB/FCS for 1 hour at room temperature, followed by three times washing in TBS and incubation with avidin–peroxidase complex (1:100 in DB/FCS; Sigma) for 1 hour. After another washing step, labeling was visualized with 3,3′-diaminobenzidine-tetra-hydrochloride (DAB, Sigma) containing 0.01% hydrogen peroxide. All sections were counterstained with Meyer's hematoxylin, dehydrated, and mounted in Eukitt (Sigma).

IL-6 expression in colon tissues was inspected using a standard immunohistochemistry method. Immunostaining was done on serial sections as described previously by Zeng et al. [11 (link)]. Briefly, the tissues were fixed in 10% formaldehyde and embedded in paraffin and then cut into 4 μm sections. Tissue sections were, respectively, deparaffinized in xylene and rehydrated by grade alcohols. The tissue was incubated overnight at 4°C with primary antibody specific for IL-6 (Santa Cruz, CA, USA, dilution 1 : 200). The tissue sections were then incubated with biotin-labeled goat anti-mouse antibody (Sigma, USA) followed by exposure to avidin-peroxidase complex (Sigma, USA). Staining was developed with diaminobenzidine (DAB, Sigma) substrate and sections were counterstained with hematoxylin.