Alexa fluor 568 coupled goat anti rabbit secondary antibody

Caco-2 monolayers were fixed in 4% paraformaldehyde for 10 min at room temperature. Cells were blocked in 5% normal goat serum in PBS for 1 h at room temperature. Then monolayers were probed with a rabbit anti-ZO-1 antibody for 12 h at 4 °C followed by incubation with Alexa Fluor 568-coupled goat anti-rabbit secondary antibody (Invitrogen, Carlsbad, CA) at room temperature in dark for 1 h. Slides were mounted and observed using fluorescence microscopy (Nikon Eclipse: TE 2000-E, Japan).

Caco-2 monolayers or HT-29 cells were pretreated with bacteria. Cells were fixed in 4% PFA for 10 min at room temperature and then blocked in 5% normal goat serum–PBS for 1 h at room temperature. Subsequently, Caco-2 monolayers were probed with rabbit anti-ZO-1 antibody (1:200) and HT-29 cells were probed with rabbit anti-MUC2 antibody (1:100) for 12 h at 4°C. The cells were then incubated with Alexa Fluor 568-coupled goat anti-rabbit secondary antibody (Invitrogen, Carlsbad, CA, USA) at room temperature in the dark for 1 h. The membranes were mounted with Fluoroshield with DAPI (4′,6-diamidino-2-phenylindole) (Sigma-Aldrich, St. Louis, MO, USA) and examined under a fluorescence microscope (Nikon Eclipse TE2000-U).