Visualization of vascular dose painting effects via immunofluorescent labeling of DNA DSBs and vascular endothelial markers.

<p><b>A.</b> Mouse brain endothelial cells co-cultured with human GBM cells (1∶7, respectively) <i>in vitro</i> show enhanced RT damage when irradiated (4 Gy) with 1 mM GNPs. Upper : Immunofluorescence imaging of γh2ax foci and DAPI in normal mouse brain endothelial cells with the indicated treatments. Lower: Quantitative analysis of γh2ax foci (yellow) for N >10 viable nuclei (blue) of normal murine brain endothelial cells co-cultured with human GBM cells <i>in vitro</i>. Error bars, 95% confidence interval. Statistical significance was determined using a one-tailed <i>t</i>-test (α = 0.05), with <i>p</i><0.05 considered significant. <b>B.</b> Brains irradiated immediately following GNP injection leads to considerable colocalization of DNA DSB and blood vessels compared to those receiving RT alone. Healthy brains were mock-irradiated or irradiated with 20 Gy (whole-brain) immediately (<5 min) after i.v. administration of 1.25 g Au/kg GNPs or saline. Mice were sacrificed 24 hours later, and their brains were fixed/stained for γh2ax, CD31, and DAPI. C. γh2ax colocalization with CD31-positive cells was performed by calculating Mander’s coefficient (M2) in binary projections of CD31 and γh2ax channels.