We used 29 human eyes from donors aged 13 to 88 years (UK Transplant Support Service, Bristol Eye Bank, Bristol, UK). Donor eyes were obtained and used in accordance with the provisions of the Declaration of Helsinki for research involving human tissue. Whole globes were dissected in a Petri dish lined with filter paper (Grade 50; Whatman, Maidstone, UK), moistened with phosphate-buffered saline, (PBS, composition: NaCl, 90 g; NaH2PO4, 13.65 g; and KH2PO4.2H2O, 2.43 g dissolved in distilled water with final volume adjusted to 1 L [pH 7.4]; Sigma-Aldrich, Poole, UK) containing 100 U/mL penicillin and 100 μg/mL streptomycin. The anterior portion of the eye was carefully removed by a circumferential incision at the pars plana, and the cornea, together with the lens, iris, and vitreous, were discarded. The posterior globe was inspected for any evidence of subretinal blood, extensive drusen, or irregular pigmentation of the RPE or any gross disease of the retina, and those exhibiting any abnormal appearance were discarded. The neural retina was gently peeled away from the underlying RPE and cut at the optic nerve head. A series of four samples were obtained from the midperiphery of each eyecup with an 8-mm trephine (Stiefel Laboratories, Buckinghamshire, UK). The RPE was carefully brushed away with a fine sable-hair brush, and the Bruch’s membrane-choroid complex was gently teased away from the underlying sclera.
The isolated preparation was mounted between the two halves of a Perspex modified Ussing chamber. Both surfaces were rinsed several times with PBS.