Vectorshield medium with dapi

Immunohistochemistry (IHC) was performed to identify neuronal nuclei using Anti-NeuN (ABN90, EMD Millipore) and FITC anti-guinea pig (706–095-148, Jackson ImmunoResearch Laboratories Inc.) (Figure 6C). Primary and secondary antibodies were diluted to 1:500 with Animal-free Blocker and Diluent, R.T.U. (SP-5035, Vector Laboratories Inc.). Slides were kept at room temperature (RT) for 1 h, then washed for three 5 min cycles in PBS with gentle rocking. Next, slides were incubated in R.T.U. buffer for 12 h at 4°C. Following removal of excess buffer, the diluted primary antibodies were distributed between slides and incubated at 4°C for 36 h. Following three 10 min PBS wash cycles, slides were stained with secondary antibodies, diluted and distributed same as before, then incubated at 4°C for 24 h. After three 10 min PBS washes excess liquid was aspirated and glass coverslips were mounted using VectorShield medium with DAPI (H-1200, Vector Labs). Slides were kept in the dark at RT for 24 h before imaging.

Ileal sections were deparaffinized, rehydrated, and boiled in antigen retrieval solution (pH 9.0) (Nichirei Bioscience) at 105°C for 20 min. Sections were blocked in 20% Block Ace (Dainippon Pharmaceutical) in PBS containing 5% goat serum (Sigma-Aldrich) at room temperature for 30 min. After blocking, the sections were incubated with primary antibodies: anti-Muc2 (1 μg/ml, sc-15334; Santa Cruz Biotechnology), anti-Crp1 (1 μg/ml, 77-R63, self-produced), anti-GRP78 (1 μg/ml, ab21685; Abcam), anti-calreticulin (10 μg/ml, #62304; Cell Signaling Technology), anti–Ephrin-B2 (10 μg/ml, AF496; R&D systems), and anti-MIST-1 (0.25 μg/ml, ab187978; Abcam) at 4°C for overnight. Then, the sections were incubated with fluorescent-conjugated secondary antibodies (Life Technologies) at room temperature for 1 h. After incubation, the sections were covered with coverslips mounted with VECTORSHIELD medium with DAPI (Vector Laboratories), sealed with nail polish and dried. Fluorescence images were observed using LSM510 Confocal Laser Scanning Microscope (Carl Zeiss).

After leaving the slides at room temperature for 1 h, they were washed with PBS in three 5 min cycles. Next, the slides were incubated in Animal-free R.T.U. Blocker (SP-5035, Vector Laboratories Inc.) overnight at 4°C. To identify astrocytes and microglia/macrophages, immunohistochemistry was performed using the primary antibodies Anti-GFAP (CPCA-GFAP, EnCor Biotech.) and Anti-Iba-1 (019-19741, Wako Chem.), respectively. Primary antibodies were diluted to 1:500 in Animal-free Blocker, R.T.U (SP-5035, Vector Laboratories Inc). The primary antibodies were then distributed across the slides and incubated at 4°C for 36 h (Figure 6C). Following three 10 min PBS wash cycles, slides were stained with secondary antibodies (Cy3 anti-rabbit, 711-165-152, Jackson Immuno.) (Cy5 anti-chicken, 703-175-155, Jackson Immuno.) diluted 1:500 in animal-free blocker and incubated at 4°C for 24 h. After three 10 min PBS washes the excess liquid was aspirated, and glass coverslips were mounted using Vector Shield medium with DAPI (H-1200, Vector Labs). Slides were kept in the dark at RT for 24 h before imaging. Image acquisition was performed on a Leica DMi8 microscope running LAS X Premium software. Peripheral components include a DFC9000 GT camera, EL-6000 light source (30 ms), HC PL APO 10x/0.45 objective, and Leica Y3 and Y5 filter cubes. Acquisitions were centered over the EDH (Figure 6C).