The 4% paraformaldehyde fixed ileum tissue samples were embedded in paraffin and sectioned in 5‐μm thickness. The following primary antibodies were incubated overnight at 4°C: anti‐mucin 2 (1:2000; Abcam, Cambridge, UK), anti‐defensin 5 (1:50; Abcam), anti‐defensin 6 (1:5000; Atlas, Bromma, Sweden), anti‐LYZ (1:100; Abcam), anti‐Occludin (1:100; Proteintech, Rosemont, IL, USA), anti‐zonula occludens 1 (anti‐ZO 1) (1:200; Abcam), and anti‐Claudin 1 (1:700; Abcam). The sections were subsequently incubated for 60 min with a biotinylated secondary antibody (1:100; Zhongshan, Beijing, China). All histological images were captured under a light microscope (Leica, Wetzlar, Germany) at 200× magnification. Image‐Pro Plus 6.0 (IPP6.0; Media Cybernetics, Inc., Rockville, MD, USA) was used for morphological analysis.
The inflammation score was determined by a blinded observer and was strictly based on a fully proven method that assigns a histological score of 0–4 to quantify the degree of inflammation. The criteria of this system include inflammatory cells, goblet cell depletion, immune infiltration, and architecture destruction.11
The inflammation score was determined by a blinded observer and was strictly based on a fully proven method that assigns a histological score of 0–4 to quantify the degree of inflammation. The criteria of this system include inflammatory cells, goblet cell depletion, immune infiltration, and architecture destruction.