The methods conformed to the Declaration of Helsinki for research involving human subjects and have Liverpool Research Ethics Committee approval. Nine surgically excised subfoveal CNV membranes secondary to AMD were obtained during pars plana microsurgery. Normal human eyes were obtained from the local eye bank (Royal Liverpool University Hospital). All specimens (globes and membranes) were fixed in formalin and embedded in wax. After the blocks were cooled, 5-μm sections were cut with a rotary microtome (model A532; Shandon, Sewickley, PA). Sections were placed on 3% aminopropylethoxysilane-coated (APES; Sigma-Aldrich, Poole, UK) glass slides before they were dewaxed in a 60°C oven for 20 minutes. They were then transferred to xylene for 5 minutes (one change) and rehydrated through 5-minute immersions in descending ratios of alcohol (99%–95%; one change of each) to distilled water before being either stained histologically (with hematoxylin and eosin) or immunohistochemically.
Immunoreactivity for monoclonal antibody (AC133-1; Miltenyi Biotec, Cologne, Germany) against a human antigen recently identified as a marker of both EPCs and HSCs and absent on mature endothelium was compared to that for cell markers specific for fully differentiated vascular endothelial cells: von Willebrand factor (vWF), CD34 and CD31 (all 1:100; Dako, High Wycombe, UK), macrophages (CD68; KP1, 1:100; Dako), and RPE (panel cytokeratins/cytokeratin 18 clone CY90 (CK18, 1:10; Sigma-Aldrich). Briefly, sections were rehydrated by washing in TBS for 10 minutes. Nonspecific binding was blocked by exposure to 5% normal goat serum (NGS; Sigma-Aldrich) in TBS (vol/vol) for 20 minutes in a humidity chamber. The blocking solution was then drained off, and the primary antibody was added overnight at +4°C at the manufacturer’s recommended dilution. After three 5-minute washes with Tris-buffered saline (TBS) and according to the manufacturer’s instructions, secondary amplification (Envision; Dako) of alkaline phosphatase (AP)-labeled polymer conjugates to affinity-purified goat anti-mouse immunoglobulins was performed with a 30-minute incubation at room temperature. After a subsequent washing in TBS (three times, 5 minutes each), immunoreactivity was visualized with fast red chromogen for 5 minutes and then sections were mounted under a glass coverslip, using mounting medium (Immunomount; Euro-Path Ltd., UK). The cell type that exhibited positive immunostaining was determined by morphologic and cytologic criteria as well as immunostaining. In negative control experiments, preimmune serum replaced the specific antibodies. All specimens were sequentially viewed in their entirety under a ×40 objective and each field of view was scored as either a fibrotic or cellular region (>50%) of the membrane. The number of cells per field were scored as 0 (−); 1 to 3 (+); 4 to 10 (++), or >10 (+++) cells per field.