Neovascular complexes of patients with PDR and nonischemic high myopia (hereafter referred to as fibrovascular membranes) were obtained from patients undergoing retinal surgery. Surgical specimens were fixed in ice-cold acetone immediately after excision. Thawed tissue sections were rehydrated with PBS and treated with PBS/1% dimethyl sulfoxide/0.3% Triton X-100/5% normal goat serum (staining buffer). Samples were then incubated overnight (4°C) with polyclonal rabbit anti–PEDF (diluted 1:500; BioProducts, Middletown, MD) antibody. This antibody was previously demonstrated to bind PEDF from different sources, as judged by Western blot analysis,
10 and it abrogated the inhibitory effects of PEDF on VEGF-A–induced phosphorylation of ERK-1 and ERK-2 in bovine retinal endothelial cells and on endothelial cell proliferation.
40 Monoclonal mouse antivimentin (diluted 1:400; Immunotech, Beckman Coulter, Krefeld, Germany), anti–human cellular retinaldehyde-binding protein (CRALBP; clone B2, 1 μg/mL; a kind gift of John C. Saari, Department of Ophthalmology, University of Washington, Seattle, WA)
41 or anti–glial fibrillary acidic protein (GFAP, diluted 1:500; clone 6F2; Dako)
42 antibodies were directed against known markers of glial Müller cells. Monoclonal antibodies were added together with anti–PEDF antibody to identify glial cells in double-staining experiments. Specimens were washed with PBS/1% BSA and then incubated with cyanogen (Cy) 2-conjugated goat polyclonal anti–rabbit IgG and Cy 3-conjugated anti–mouse IgG (Dianova, Hamburg, Germany; both diluted in staining buffer). Then samples were counterstained (Hoechst 33342, 5 μg/mL; Sigma), covered by a nonfluorescent sealant and were examined under a fluorescent microscope (Axioskop; Carl Zeiss, Oberkochen, Germany) equipped with a digital camera. A scoring system implicating staining intensity was used for determining PEDF expression in membrane derived from patients with diabetes and patients with myopia, respectively. Four categories of staining were defined: no (0), faint (1), modest (2), and intense (3) stain. The intensity of staining in each of these categories was assessed by the authors (YY, JL, WE) without knowledge of the clinical or pathologic data for the particular sample.