Postmortem human eyes were obtained from the Manchester Eye Bank after removal of the corneas for transplantation. In all cases, prior consent had been obtained for the ocular tissue to be used for research, and guidelines established in the Human Tissue Act 2004 were followed. Our research adhered to the tenets of the Declaration of Helsinki. Eyes were from adult human donors without known retinal disease or visual impairment.
Ten globes (five pairs from five donors, aged between 60 and 83 years) were dissected, removing the iris, lens, and vitreous body, to obtain the neurosensory retina and the tissue complex that includes the RPE, Bruch's membrane, and choroid (henceforth known as Bruch's/choroid complex). Ten further globes (five pairs from another five donors, aged between 63 and 75 years) were dissected to obtain isolated Bruch's membrane, by removing the RPE and the choroid through repeated application of a cell scraper until a homogeneous grey tissue layer was left.
The tissues described above were pooled (i.e., each from 10 globes) to produce three samples as follows: Bruch's/choroid complex, Bruch's membrane, and neurosensory retina. These tissue samples were cut up separately into fine pieces. PGs were solubilized from each of the three samples using guanidinium chloride extraction. Briefly, each sample was added to 20 mL 4 M guanidine HCl, 0.5 M NaOAc, and 0.5 mL protease inhibitor cocktail (AEBSF, aprotinin, bestatin, EDTA, E-64, and pepstatin [Sigma, Poole, UK]) at pH 5.8, and left for 18 hours at 4°C on a rotating mixer. Each sample then underwent centrifugation, and the supernatant was dialyzed twice (1:50 volume, for 16 hours at room temperature) against the DEAE starting buffer: 8 M urea, 50 mM Tris HCl at pH 6.8. Samples were filtered with a 0.22-μm filter prior to use. Anion exchange chromatography was performed separately for the three samples using a 1.6 mL DEAE Sepharose Fast Flow column (GE Healthcare Life Sciences, Little Chalfont, UK), equilibrated with 5-column volumes of 50 mM Tris HCl at pH 6.8, followed by a gradient of 0 to 1 M NaCl in 50 mM Tris HCl, pH 6.8, over 13-column volumes; the fractions collected were each of volume 500 μL.
Chromatography fractions were probed with Alcian blue (Sigma) using the critical electrolyte method
20 in order to identify those fractions with highest concentration of PGs. Here, 100 μL of each fraction was applied to a nitrocellulose membrane under vacuum in a slot blot. The membrane was blocked by submersion for 1 hour in 200 mL 1% (w/v) bovine serum albumin in PBS (Oxoid; 137 mM NaCl, 2.6 mM KCl, 8.2 mM Na
2HPO
4, 1.5 mM KH
2PO
4, pH 7.3) at room temperature on a mixing tray. Following this, the membrane was submerged in 200 mL Alcian blue (0.2% [w/v] at pH 2.5 in PBS) for 20 minutes, and destained by submersion in 0.05 M MgCl
2, 3% (v/v) acetic acid, with three changes for 10 minutes each. The nitrocellulose membrane was dried and the degree of Alcian blue staining was analyzed using densitometry measurements.