Immunofluorescence Imaging of Intestinal Tight Junctions

We applied the methods which usually utilized and described in previous articles [19 (link)]. Frozen sections of ileum were prepared for detecting ZO-1 morphology. After preparatory work, the sections were incubated with monoclonal rabbit antibody against zonula occludens (ZO)-1 (Invitrogen, Carlsbad, CA) diluted at 1:200 in 5% normal goat serum in phosphate buffer saline (PBS) at 4 °C overnight. Then the sections were incubated with secondary Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody (Invitrogen) at 1:200, Alexa Fluor 594-conjugated phalloidin (Invitrogen) at 1:100, and diamidine-2-penylindole (DAPI) (Sigma) at 1:1000 for 1 h at room temperature. Images were obtained using a TCS SP5 laser confocal microscopy (Leica, Germany).