Single B6-RPE07 cell suspension (1 × 104) were seeded in 8-chamber slides (Nalge Nunc International, Rochester, NY) and were cultured for 4 days. Confluent B6-RPE07 cells were washed briefly with cold PBS and then fixed with 100% ice-cold methanol (Fisher Scientific) for 10 minutes. After thorough washing, samples were permeablized with 0.5% (vol/vol) Triton X-100 in Tris-buffered saline (TBS) for 5 minutes at room temperature. Samples were incubated with rabbit anti–ZO1 (1:100 in TBS; Zymed, South San Francisco, CA), goat anti–β-catenin (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti–claudin-1 (1:100; Stratech Scientific Ltd., Newmarket, Suffolk, UK), rabbit anti–claudin-3 (1:100; Zymed), mouse anti–occludin-1 (1:100; Zymed), rabbit anti–pan cytokeratin (1:50; ICN Pharmaceuticals, Costa Mesa, CA), mouse anti–cytokeratin 19 (1:50; DAKO, Glostrup, Denmark), rabbit polyclonal anti–cytokeratin 12 (1:50; Santa Cruz), rabbit anti–GFAP (1:100, Abcam, Cambridge, UK), rabbit anti–integrin β5 (1:100; Abcam), biotinylated anti–mouse CD36 (1:100; Biolegend, San Diego, CA), or biotinylated anti–mouse CD51 (integrin αv, RMV-7, 1:100; BD Biosciences, Oxford, UK) for 1 hour. After washing, samples were incubated with fluorescein isothiocyanate (FITC)–conjugated goat anti–rabbit immunoglobulin (1:200; Zymed), goat anti–mouse immunoglobulin (1:200; Zymed), or donkey anti–sheep/goat immunoglobulin (1:100; Serotec, Oxford, UK), or FITC-conjugated streptavidin (1:200; BD Biosciences) and propidium iodide (PI; 1:200; Molecular Probes, Eugene, OR) for another hour. After thorough washing, the immunofluorescence-stained samples were mounted with mounting medium (Vectashield; Vector Laboratories, Burlingame, CA) and examined under a fluorescence microscope (LSM5110 META; Carl Zeiss, Göttingen, Germany).
To determine the apical Na+/K+-ATPase and basal bestrophin expression in polarized B6/RPE cells, a single-cell suspension of B6-RPE07 was seeded in 6-mm cell culture inserts (0.4-μm pore size; Corning, Fisher Scientific) in serum-free epithelial medium (Quantin 286; PAA Laboratories Ltd.) for 4 to 5 days. After the cells had reached confluence, insert membranes were fixed in ice-cold 100% methanol for 10 minutes. Membranes were permeabilized with 1% Triton X-100 for 5 minutes and were blocked with 5% BSA for 30 minutes. Rabbit anti–Na+/K+-ATPase (1:100; Santa Cruz Biotechnology) or rabbit anti–mouse bestrophin (1:100; Abcam) was added to the membrane and incubated at room temperature for 1 hour, followed by FITC-conjugated goat anti–rabbit immunoglobulin (1:200; Zymed) and PI for another hour. After thorough washing, the membranes were mounted with mounting medium (Vectashield; Vector Laboratories) and examined by confocal microscopy.