Fig. 1

RFC1 protein is abundantly expressed in endothelial cells and pericytes of the retinal microvessels. A Ex-vivo labeling of 20 µm radial cryo-section from PFA fixated eyeballs of naive Swiss Albino mice with anti-RFC1 antibody (red). Abundant RFC1 immunopositivity is observed along ganglion cell layer (GCL), inner plexiform layer (IPL), outer plexiform layer (OPL) where retinal microvessels forms vascular horizontal vascular beds; as well as the retinal pigment epithelium (RPE). However, this preparation limited the observation of microvessels, hence inner BRB, as it commonly included microvessels circularly rather than longitudinally. Scale bar= 25 µm B RPE which is known to express RFC1 previously is well stained with anti-RFC1 antibody (red) as our positive control. C The microvessels constituting inner BRB form the deep vascular plexus of the retina were immunohistochemically labelled with anti-RFC1 antibody in PFA fixated whole-mount retinas. D Retinal microvessels which were obtained via retinal trypsin digestion method that allowed to get only microvessels (< 9 μm diameter) were immunofluorescently labeled with anti-RFC1 antibody (n = 6 retina; red). Nuclei were labeled with Hoechst 33258 (blue) in all the rows. E The endothelial marker CD31 (green) (n = 3). The “bump-on-a log” shaped pericyte body was positively stained with anti-RFC1 antibody, but negative for endothelial marker CD31. Hence, RFC1 staining was specific. F–H RFC1 (red) also colocalized with accepted pericyte markers NG2, CD13, PDGFR-β (green) shown respectively (n = 3/marker). Nuclei were labeled with Hoechst 33258 (blue). Scale bars =10 μm