6. A 1:200 dilution of primary zonula occludin (ZO-1) rabbit antibody was prepared in 3% (w/v) BSA in PBS, and 400 was added to each apical compartment and incubated at 4°C μL in a damp box (containing damp tissue to reduce evaporation) overnight. 7. The following day, the epithelia were washed twice with 200 PBS and labelled using μL 400 of a 1:1000 dilution of Alexa Fluor green (AF488) in PBS, applied to the apical μL surface of each epithelium for 1 hour at room temperature in the dark. 8. Afterwards, epithelia were again washed twice with 200 PBS, and 400 of 5 μL μL μM Hoechst in PBS was applied to label cell nuclei and incubated for 15 minutes at room temperature in the dark. 9. Afterwards, the epithelia were washed 3 x 200 PBS, and the cell culture inserts were μL cut from their plastic holders using a scalpel and placed cell-side-up on a microscope slide. One drop of mounting media was applied under a cover slip, which was then varnished onto the slide. The slides were left to dry overnight in the dark (wrapped in foil) at 4°C before visualisation on a confocal microscope at the appropriate wavelengths.
6. A 1:200 dilution of primary zonula occludin (ZO-1) rabbit antibody was prepared in 3% (w/v) BSA in PBS, and 400 μL was added to each apical compartment and incubated at 4°C in a damp box (containing damp tissue to reduce evaporation) overnight. 7. The following day, the epithelia were washed twice with 200 μL PBS and labelled using 400 μL of a 1:1000 dilution of Alexa Fluor green (AF488) in PBS, applied to the apical surface of each epithelium for 1 hour at room temperature in the dark. 8. Afterwards, epithelia were again washed twice with 200 μL PBS, and 400 μL of 5 μM Hoechst in PBS was applied to label cell nuclei and incubated for 15 minutes at room temperature in the dark. 9. Afterwards, the epithelia were washed 3 x 200 μL PBS, and the cell culture inserts were cut from their plastic holders using a scalpel and placed cell-side-up on a microscope slide. One drop of mounting media was applied under a cover slip, which was then varnished onto the slide. The slides were left to dry overnight in the dark (wrapped in foil) at 4°C before visualisation on a confocal microscope at the appropriate wavelengths.