N/N 1003A rabbit lens epithelial cells were grown to confluence for 4 days on a 0.4-μm pore size polyester membrane in 6.5 mm diameter wells (Transwell; Corning, Inc.), in DMEM+FBS in humidified atmosphere containing 5% CO2 at 37°C. The medium was refreshed on day 2. On the fourth day, the membranes on which the cells had grown were cut from the Transwell insert, washed in phosphate-buffered saline (PBS: 150 mM NaCl, 25 mM NaH2PO4 [pH 7.4]) and fixed at room temperature for 30 minutes in 2% (vol/vol) paraformaldehyde in PBS (Electron Microscopy Sciences, Hatfield, PA). After three washes in PBS, the cells were permeabilized in 0.1% (vol/vol) Triton X-100 in PBS for 30 minutes at room temperature. After three washes of 5 minutes each in PBS, the cells were incubated for 1 hour in a solution containing 3% (vol/vol) normal goat serum (Biomeda Corp., Foster City, CA) and 3% (wt/vol) BSA (United States Biochemical Corp., Cleveland, OH) at room temperature. The cells were then labeled with a mouse antibody raised against the tight junction marker ZO-1 (1:200 in PBS; Zymed, South San Francisco, CA) for 2 hours at room temperature. Cells were washed three times in PBS and incubated with an Alexa 594-labeled goat anti-mouse antibody (1:200; Invitrogen-Molecular Probes, Eugene, OR) for 1 hour at room temperature. Finally, the cells were washed in PBS before labeling with a nuclear stain (TO-PRO 3, 1:1000; Invitrogen-Molecular Probes). The membranes were mounted on glass slides (Fluorosave; Calbiochem, San Diego, CA) and observed with an inverted confocal microscope (LSM 510; Carl Zeiss Meditec GmbH, Göttingen, Germany).