Anti claudin 1

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

Immunofluorescence (IF) was performed to validate claudin-1 expression ex vivo. The distal colon was divided, opened longitudinally, and rinsed with cold PBS. The tissues were fixed in 10% neutral-buffered formalin for ∼48 hours and then transferred into 70% ethanol. The tissues were processed for routine histology (H&E). Deparaffinization and antigen retrieval were performed on adjacent, unstained sections, as described previously (14 (link)). For peptide staining, the sections were blocked with 2% bovine serum albumin (BSA) for ∼1 hour and 0.5 μM of peptide for 10 minutes. For antibody staining, blocking was performed using 10% normal serum followed by ∼1 hour of incubation with either anti–claudin-1 (#15098; Abcam, Cambridge, MA) or anti–β-catenin antibody (#16051; Abcam, Cambridge, MA). Goat anti-rabbit IgG antibody conjugated with either AF488 (#A-11029; Life Technologies, Carlsbad, CA) or AF647 (#205-605-108; Jackson, West Grove, PA) was used. For colocalization of RTS*-Cy5.5 and anti–claudin-1 antibody staining, the tissues were incubated with peptide first for 10 minutes followed by goat anti-rabbit IgG antibody conjugated with AF488 (#A-11029; Life Technologies, Carlsbad, CA). Fluorescence images were collected using an inverted confocal microscope (Leica inverted SP5).