Ds cooled camera head

The BEECs were plated on glass coverslips in a 24-well flat-bottomed culture plate, and the treated BEECs were washed with PBS three times, incubated with a mitochondrial localization probe (200 nM Mito-Tracker Red CMXRos, Beyotime Biotechnology, C1049B) according to the manufacturer’s instructions, and then fixed with 4% paraformaldehyde for 15 min on ice. The cells were rinsed three times with PBS and then incubated for 60 min in a blocking solution (1 × PBS/5% goat serum/0.3% Triton X-100) to reduce the nonspecific background. After being washed with PBS three times, the cells were incubated with goat anti-rabbit IgG (H+L) and FITC conjugate (TRANSGEN, Beijing, China) for 1 h at room temperature and DAPI (Solarbio, Beijing, China) for 10 min at 37 °C. The cells were then covered with a coverslip, and the edges were sealed to prevent drying. The cells were observed and photographed using a Nikon Eclipse Ti-U inverted fluorescence microscope equipped with a Nikon DS cooled camera head (Nikon, Hitachi, Tokyo, Japan).

Attachment of F4⁺ ETEC/VTEC/EPEC to ileal mucosa was determined by an indirect immunofluorescence assay. Ileal tissue samples fixed with 4% paraformaldehyde were embedded in paraffin, cut into 4-μm sections, and collected on silanized slides. The sections were then incubated with rabbit anti-F4 fimbriae antiserum (China Institute of Veterinary Drug Center, Beijing, China) overnight in a humidified chamber at 4 °C. Goat anti-rabbit Cy3-conjugated (AP307F; Sigma-Aldrich) was used as secondary antibody, and DAPI 3 (Sigma-Aldrich) was used for nuclear staining. The slides were visualized and photographed using a Nikon Eclipse Ti-U inverted fluorescence microscope equipped with a Nikon DS cooled camera head (Nikon).

IPEC-J2 cells seeded on glass coverslips were infected with mCherry-S. Infantis (3 × 10⁸ CFU), as described above. After the growth medium containing 50 μg/ml of NAL was removed, the cells were washed, fixed with 4% paraformaldehyde for 15 min on ice, permeabilized with 0.2% (vol/vol) Triton X-100 (Sigma-Aldrich, Saint Louis, MO) and blocked with 1% bovine serum albumin. Subsequently, the cells were incubated with mouse anti-cytokeratin-18 primary monoclonal antibody at a dilution of 1:200 (Ab668; Abcam, Cambridge, UK) for 45 min at 4°C, following by secondary antibody goat anti-mouse fluorescein isothiocyanate-conjugated IgG (F4143; Sigma-Aldrich). The cell nuclei were stained using 4',6'-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). The recovered S. Infantis in the liver from SI pigs were determined by staining frozen sections of liver with the rabbit anti-pig α-tubulin primary monoclonal antibody (1:200 dilution, ab52866, Abcam), secondary antibody goat anti-rabbit FITC-conjugated IgG (F6005, Sigma-Aldrich) and DAPI. The coverslips and slides were visualized and photographed under an inverted Nikon Eclipse Ti-U fluorescence microscope equipped with a Nikon DS cooled camera head (Nikon, Tokyo, Japan).