Immunofluorescence Analysis of Ileal Tissue

The ileal tissues from the distal portion of the ileum were freshly isolated and paraffin-embedded after fixation with 10% neutral buffered formalin. Immunofluorescence was performed on the paraffin-embedded sections (5 μm). After preparation of the slides, as described previously (31 (link)), the tissue samples were incubated with the indicated primary antibody, anti-lysozyme (1:100, sc27958, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), at 4°C overnight. The samples were then incubated with the sheep anti-goat Alexa Fluor 594 (A11058, Life Technologies, Grand Island, NY, USA) and DAPI (D1306, Life Technologies, Grand Island, NY, USA) for 1 h at room temperature. The tissues were mounted with SlowFade (s2828, Life technologies, Grand Island, NY, USA), followed by a coverslip, and the edges were sealed to prevent drying. The specimens were examined with a Zeiss laser scanning microscope 710 (Carl Zeiss, Inc., Oberkochen, Germany).