Analysis of the VEGF-A pathway in the two cases. (A) mRNA expression levels of vegf-a, flt-1, kdr, hif-α, herg1a and herg1b in the RB tumor mass. (B) Amount of VEGF-A released in the vitreous fluid by case 1 and case 2. The medium was collected and used for VEGF-A measurement as reported in Methods section. Data are reported as mean ± ESM. Median value relative to control group (as described in 22) is reported (see dotted line). (C) H&E staining (left panels) and immunohistochemistry for VEGF-A (middle panels) and hERG1 proteins (right panels) on case 1 and 2. Inset: magnification of the picture reported in the corresponding panel. Scale bar = 100 μm; Inset scale bar = 50 μm (D) Immunostaining for VEGF-A and hERG1, analysis of microvessel density (MVD) and evaluation of apoptotic and proliferation index (AI and PI respectively). The intensity of VEGF-A and hERG1 staining was also evaluated in normal retinal tissue and the percentage of positive cells was 12,2 ± 2% and 23,08 ± 8% respectively.