Intestinal tissues of mice were embedded in a frozen section compound (Surgipath FSC22, Leica Microsystems, Wetzlar, Germany). Thin sections (10 µm) were fixed in 4% paraformaldehyde, blocked in 1% BSA in PBS, and incubated for 2 h at room temperature with an anti-TLR4 mouse monoclonal antibody or an anti-villin rabbit polyclonal antibody with gentle rocking. After washing 3 times with PBS, appropriate fluorescent-conjugated secondary antibodies were used for visualizing the antigen. Nuclei were visualized by staining with 1 μM 4′,6′-diaamidino-2-phenylindole (DAPI) in PBS for 10 min. Confocal images were acquired on a ZEISS LSM 880 confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany). For z-stack analysis, images were obtained along the z-axis at 0.3-μm intervals and subjected to three-dimensional analysis using ZEN software (Blue edition, 2.3, Carl Zeiss).