Carl 710 confocal microscope

Par‐C10 cell clusters grown on GFR‐MG were fixed in 4% paraformaldehyde for 10 min at room temperature, incubated with 0.1% Triton X‐100 in PBS for 5 min, and washed three times with PBS. The cells were then incubated with 5% goat serum for 1 h at room temperature and washed three times with PBS. This was followed by incubation overnight at 4°C with rabbit anti‐ZO‐1 (Invitrogen, Carlsbad, CA) at 1:200 dilution in 5% goat serum. The next day, cells were allowed to warm up to room temperature for 20 min and washed three times for 5 min with PBS. Next, cells were incubated for 1 h with Alexa Fluor 488‐conjugated goat anti‐rabbit (1:500 dilution in 5% goat serum) and washed three times with 1 PBS. Finally, cells were stained for 1 h with phalloidin to localize filamentous actin (1:400 dilution in PBS; Sigma Aldrich, St. Louis, MO), and 5 min with TO‐PRO‐3 iodide (1:10,000 dilution in PBS; Thermo‐Scientific, Waltham, MA) to visualize nuclei. Images were obtained using a Carl Zeiss 710 confocal microscope (Carl Zeiss, Oberkochen, Germany). All images were analyzed using ZEN 2012 (Blue Edition; Carl Zeiss) and Graphpad Prism 6 (GraphPad Software, Inc. La Jolla, CA) software. Lumen sizes (diameter of the lumen) were measured at the maximum diameter of 10–15 cell clusters per experimental group.