For immunohistochemistry and polarization studies, cells were grown for 18 days at confluence in 24-well plates containing one circle coverslip of glass (12-mm diameter) (Thermo Scientific, Menzel-Gläser; Braunschweig, GE) inside each well. Cells were washed with PBS and fixed with methanol (ZO-1 and claudin-1) or paraformaldehyde (FN and Coll IV) for 10 minutes, washed again with PBS twice, and blocked with 2% BSA and 0.05% Tween in PBS overnight at 4°C. Mouse anti-ZO-1, rabbit anti-claudin-1 (Zymed Laboratory Gibco, Invitrogen, San Diego, CA), rabbit anti-FN, rabbit anti-Coll IV (Abcam, Cambridge, MA), and mouse anti-N+/K+ ATPase (Millipore), all diluted to 1:200, were incubated for 1 hour at room temperature (RT). After washing with PBS, cells were further incubated with Alexa 488 goat anti-rabbit and Alexa 594 donkey anti-mouse secondary antibodies (Invitrogen) for 1 hour at RT. After washing with PBS, the slides were mounted with mounting medium containing DAPI for fluorescence (Vectashield; Vector Laboratories, Burlingame, CA). Images were acquired with a confocal laser scanning microscope (FV1000; Olympus, Hamburg, Germany).