CCR2 Blocker Inhibits Choroidal Neovascularization Figure 3. VEGF expression. (A) Immunohistochemistry of macrophages (green) and VEGF (red) in cryosections on Day 3. Significantly higher levels of VEGF were expressed in macrophages at the photocoagulated sites. VEGF localized mainly in infiltrating macrophages at the laser injury sites. INCB3344 apparently decreased VEGF immunoreactivity compared to vehicle treatment. The negative control omitting the primary antibody (second antibody only) had detectable auto-fluorescence in RPE. Scale bar = 100 mm. (B) VEGF protein levels in the choroid-RPE were quantitatively measured by ELISA. VEGF levels on Day 3 were significantly suppressed by INCB3344 treatment. (n = 8, *P = 0.012). (C) The expression of VEGF mRNA derived from macrophages isolated from choroid-RPE complexes was detected by real-time PCR on Day 3 after photocoagulation. The increased VEGF mRNA expression in infiltrating macrophages was significantly suppressed by INCB3344 treatment (**,***P,0.001, n = 3). doi:10.1371/journal.pone.0028933.g003 PLoS ONE | www.plosone.org 6 December 2011 | Volume 6 | Issue 12 | e28933