PDGF-BB–dependent Shh expression in VEGF-mediated corneal angiogenesis model. VEGF-containing (5 µg) pellets were implanted into the cornea of C3HeB/FeJ mice (n = 8 per group). (A) Five days later, the Shh mRNA level was evaluated by RT-qPCR in Ctrl and VEGF-treated corneas with or without the PDGFR-β inhibitor imatinib (20 nmol); β-actin was used as a housekeeping reference gene. Data of RT-qPCR represent means ± SD of relative expression vs control corneas. **P < .005; ***P < .0005. (B) Shh (green, Alexa 488) and NG2 (red, Alexa 568) expression was detected by immunostaining and imaged with a Zeiss Observer Z1 fluorescence microscope. (C-D) Shh (green, Alexa 488), EC (CD31, red, Alexa 568), and NG2 (pink, Alexa 647) expression was revealed by immunohistology. Nuclei were counterstained using 4,6 diamidino-2-phenylindole (DAPI; blue). Images were shot with an Olympus FV1000 confocal microscope at low (×20) (C) or high (×40) (D) magnification, respectively. In D, far-right panel shows isosurface analysis by Imaris Bitmap software.