Figure 2. Histopathological characterization of FVM tissue from patients with severe PDR. Light micrographs of the control retina (A–E) and fibrovascular membranes (FVMs) from five different cases (F–DD) processed for immunohistochemistry using primary antibodies against CD 31 (A, F, K, P, U, Z), smooth muscle actin (SMA, B, G, L, Q, V, AA), glial fibrillary acidic protein (GFAP, C, H, M, R, W, BB), retinal pigment epithelium-specific protein 65 (RPE65, D, I, N, S, X, CC), and bestrophin-1 (BEST-1, E, J, O, T, Y, DD; all in red) and counterstained with hematoxylin (blue). In the control retina, faint CD31 staining of endothelial cells is observed (A), SMA localization is restricted to vessels (B), GFAP staining localizes to cells surrounding the blood vessels within the nerve fiber layer (NFL, C), and RPE65 and BEST-1 localize to the RPE cells (D, E). For FVMs derived from different patients, however, there was notable variability in the localization patterns and the number of positive cells for the different markers. All FVMs have CD31-positive cells, which are perivascular (F, K, P, U, Z). SMA-positive cells were also detected in all FVMs; some SMA staining was localized predominately to cells surrounding the vessels (G, AA) whereas other SMA-positive cells appear to be stromal (L, Q, V), located throughout the fibrous tissue of the FVM. GFAP-positive cells are also observed in all FVMs; some GFAP-positive cells are seen surrounding vessels (H, BB) whereas other GFAP-positive cells are located within the stroma of the FVM (M, R, W). Whereas all FVMs are RPE65-negative (I, N, S, X, CC), BEST-1 positive cells are distributed throughout the FVM tissue, as well as localized to perivascular cells (J, O, T, Y, DD). Scale bar=100 µm.