RPE monolayers grown on Transwell filters [31 (link)]) were fixed in 4% PFA, and subsequently permeabilized with 0.1% Triton-X 100 for 15 min. Samples were blocked in 5% normal goat serum followed by incubating with either OGC (1:100) and DIC (1:100) rabbit polyclonal antibodies overnight at 4 °C. The cells were washed and incubated with fluorescein conjugated secondary antibody (Vector Labs, Burlingame, CA, USA) for 30 min at room temperature. Transwell membranes were cut and removed from the inserts with a fine razor and mounted on a microslide. The specimen was viewed on an LSM 770 laser-scanning microscope and (Carl Zeiss, Thornwood, NY, USA).
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