Journal: European journal of ophthalmology

Article Title: VEGF Trap R1R2 suppresses experimental corneal angiogenesis

doi:

Figure Lengend Snippet: VEGF expression correlated with vascular progression in the cornea. Vascular progression was induced by bFGF pellet implantation on days 1 (A), 4 (B), and 7 (C). Vascular endothelial cells were visualized by fluorescein-conjugated tomato lectin, and the progression of vessels was visualized by the VEGF-LacZ expression on days 1 (D), 4 (E), and 7 (F). LacZ expression (in red) was observed starting at day 4 and showed greater expression at day 7 (E–F). bFGF implanted corneas were divided into 6 segments as illustrated in (G) and were analyzed by Western blot analysis (H). Fifteen kDa bands (H, lanes 1 to 6) corresponding to VEGF expression in different segments (G, lanes 1 to 6) were observed. Recombinant VEGF was used as control (H, lane 7). The highest amount of VEGF was seen close to the pellet (H, lane 3) and in the segments adjacent to the pellet (H, lanes 2 and 4). There is also a lighter band noted at the limbal area closer to the pellet (H, lane 1). No VEGF expression was observed in segments away from the pellet (H, lanes 5 and 6). bFGF implanted corneas were coimmunostained with anti-VEGF antibody (I) and macrophage marker F4/80 antibody (J; merged image (K)).

Article Snippet: Mice were then sacrificed and whole eyes were harvested and fixed in 10% neutral, buffered formalin for 24 h. The corneas were dissected and placed in blocking solution (1% BSA) for 4 h. The corneas were then incubated with a 1:200 dilution of biotin-conjugated IgG fraction of anti-β-galactosidase antibody (Rockland Immunochemicals Research Inc, Gilbertsville, PA) overnight, rinsed in PBS, and incubated with a 1:1000 dilution of rhodamine-conjugated streptavidin (Rockland) for 2 h. The specimens were rinsed in PBS and mounted on glass slides with Vectashield mounting medium for fluorescence imaging (Vector Laboratories, Burlingame, CA).

Techniques: Expressing, Western Blot, Recombinant, Control, Marker