2.11. Immunofluorescence of Zonulin and Occludin

Caco-2 cells grown until differentiation on Lab-Tek chamber slides (Nunc, Merck Millipore, Darmstadt, Germany) were treated for 3 h with 1 mg/mL PT-gliadin or PT-gliadin+bact in complete medium, then were washed with PBS and fixed with 2% formaldehyde for 15 min at room temperature. Cells were permeabilized with 0.1% triton, exposed to blocking buffer (5% BSA in PBS) for 30 min, and then incubated for 2 h at room temperature with rabbit polyclonal anti-zonulin (ZO-1) (1:100 dilution) or anti-occludin (1:100) antibody (Thermo Fisher, Waltham, MA, USA) in 1% BSA in PBS. After washing, chambers were exposed for 1 h to the secondary goat anti-rabbit antibody Alexafluor 488 (1:100 dilution) (Abcam, Cambridge, UK). Nuclei were stained for 1 min with 1 µg/mL of 4′,6-diamidino-2-phenylindole (DAPI) in saline solution, and the coverslips were mounted with Mowiol. Fluorescence signal was analyzed by recording stained images using an AxioObserver inverted microscope, equipped with the ApoTome System (Carl Zeiss Inc., Oberkochen, Germany). Microscopy imaging was performed using the Axiovision software (Zeiss).