Goat anti mouse iga

Feces were collected from Sox8^+/+ and Sox8^−/− mice. Freeze-dried feces were homogenized in PBS with EDTA-free protease inhibitor cocktail (Complete EDTA-free; Roche) and centrifuged at 4°C. Fecal supernatants were diluted in 2% BSA/PBS. MaxiSorp plates (Thermo Fisher Scientific) were coated with goat anti-mouse IgA (Bethyl Laboratories) for 1 h. The plates were washed five times with 0.1% Tween20 in Tris-buffered saline (TBS-T) and then blocked with 2% BSA/PBS at room temperature for 30 min. The plate was incubated with the diluted samples at room temperature for 1 h. After washing five times with TBS-T, the plate was incubated with HRP-conjugated goat anti-mouse IgA antibodies (Bethyl Laboratories) at room temperature for 1 h. After washing five times with TBS-T, the plate was incubated with 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific). The reaction was stopped by adding 1.2 M sulfuric acid. The absorbance was measured at 450 nm.

The concentrations of IgA were determined by ELISA. In brief, 96-well plates (MaxiSorp; Thermo Fisher Scientific) were coated with 50 µl of 10 µg/ml goat anti-mouse IgA (Bethyl
Laboratories, Montgomery, TX, USA) for 1 hr at 22–25°C. After washing with phosphate-buffered saline (PBS) containing 0.05% (w/v) Tween 20, the wells were blocked with 300 µl PBS containing
1% bovine serum albumin (Sigma-Aldrich) at room temperature for 1 hr, and 50 µl of the diluted samples was added to each well and incubated at room temperature for 1 hr. After washing, 50 µl
of 25 ng/ml goat anti-mouse horseradish-peroxidase-conjugated IgA (Bethyl Laboratories) was added, and the wells were further incubated at room temperature for 1 hr. After washing, 50 µl of
the TMB microwell peroxidase substrate system (SeraCare, Milford, MA, USA) was added and incubated at room temperature. After the addition of 25 µl of 1 M sulfuric acid, the absorbance at
450 nm was measured using an infinite F200 microplate reader (Tecan, Zürich, Switzerland). Purified mouse IgA (κ isotype control; BD) was used as a standard.
The concentrations of IL-6 were determined by ELISA using a Mouse IL-6 DuoSet ELISA (R&D Systems, Minneapolis, MN, USA).