sEphB4 inhibits angiogenesis in murine Matrigel and corneal micropocket assays. (A) Matrigel solution was injected subcutaneously into Balb/C nu/nu mice. After 6 days, plugs were removed and processed in paraffin. Individual sections were stained with Masson trichrome and representative photographs at × 20 magnification from triplicate plugs in 2 independent experiments are shown. Top left shows section of a Matrigel plug without any added proteins; top right, section of plug containing VEGF only; bottom left, section of plug with sEphB4 and VEGF; and bottom right, section of plug with CF2 and VEGF. Photomicrographs were taken with a Nikon Coolpix 5000 camera on a Nikon Eclipse E400 microscope with a 4 ×/0.13 NA objective and a 10 × eyepiece. (B) Quantitation of vascularized area averaged (± SEM) from all plugs (Scion Image software). (C) Immunofluorescent staining of dissected plugs (with or without sEphB4) with PECAM, EphB4, and EphrinB2 antibodies. (D) sEphB4 was added to Hydron polymer and sucralfate with or without bFGF and inserted into a micropocket in corneas of Balb/C mice in triplicate. Shown is neovascular response on day 4 after implantation: Hydron with bFGF (left), with no growth factor added (center), and growth factor and sEphB4 (right). (E) Quantitation of vascularization averaged (± SEM) over all corneas in each group.