Quantitative Immunohistochemical Analysis of Mucosal Mast Cells

Immunohistochemical staining was performed according to the procedure described in previous reports [21 (link), 31 (link)–33 ]. Briefly, one hour after the post-OIT non-heated OVA challenge, the proximal colon was excised, fixed by immersion in 4% paraformaldehyde and then embedded in OCT compound. Cutted sections (30 μm) were exposed to antiserum against mMCP-1, a marker of mouse mucosal mast cells (1:5,000; Moredun Scientific, Scotland, UK) and then incubated with Cy3-conjugated sheep anti-donkey IgG (1:200; Jackson Immunoresearch Laboratories). The immunostained sections were examined using a fluorescence microscope (IX71 system; Olympus, Tokyo, Japan) with a U-MWIG3 filter set (Olympus) and photographed using an Olympus digital camera (DP70; Olympus). The histological staining was quantitatively analyzed using ImageJ software.