Figure 4
BacFLT-GFP directs endothelial-specific gene expression into the retina vasculature. 10 μl of BacFLT-GFP virus solution (approximately 1 × 107 PFU of viral particles) or vehicle (PBS) were injected into the rat eye vitreous chamber. Three days after the virus injection, the rats were sacrificed and the retinas processed for immunohistochemistry using an antibody against von Willebrand factor (red) and DAPI (blue) to highlight nuclei (see Methods). Representative retinas from eyes injected with 1 × 107 pfu of BacFLT-GFP (a, b, c, d) or vehicle (PBS) (e, f, g, h). The same sections were evaluated by phase-contrast microscopy (a, e) and by anti-von Willebrand factor antibodies coupled to TRITC (red) and GFP green fluorescence. Merge images illustrate the colocalization of red and green fluorescence (in yellow) in capillary-like structures (big white rectangles in c, d, g) obtained from the areas in small white rectangle insets from b, f. Confocal three-dimensional reconstruction images of retinas injected with vehicle (h) or with BacFLT-GFP (i). In red is shown the localization of von Willebrand factor (red) within characteristic blood vessel structures. The colocalization of red (vWF) and green (GFP) fluorescence is shown in yellow (white arrowheads in i). (j) Percentage of vWF and GFP pixel co-localization in retinas injected with BacFLT-GFP, (n = 10 animals). Abbreviations: RPE, retinal pigment epithelium GCL, ganglion cell layer; INL, inner nuclear layer; and ONL, outer nuclear layer; ILM inner limiting membrane.