Occludin immunoreactivity correlated with the permeability of Evans blue at the tuberal region of the hypothalamus. A: Low magnification photomontage showing fluorescence after intravenous injection of Evans blue dye (grey). B: Low magnification photomontage of the same section showing occludin (tight junction protein, green) and MECA 32 (fenestrated capillary marker, red) immunoreactivities. Occludin (green) is expressed by all cells surrounding the third ventricle but the pattern of expression is not uniform. C–E: High magnification images captured from the same section. Cuboidal ependymal cells (C) and tanycytes at the level of the median eminence (E) display a honeycomb pattern of expression for occludin (green, empty arrows). Tanycytes at the level of the arcuate nucleus exhibit an unorganized pattern of expression for occludin (green, empty arrowhead, D). F: Color-combined image of A and B. When injected into the blood, the dye is stopped by tight junctions of the blood-brain barrier, and is confined into brain vessels that are immunoreactive for occludin (green, arrows). By contrast, the dye reaches the median eminence parenchyma through the fenestrated portal vessels that are immunoreactive for MECA 32 (red, asterisks). G: Low magnification photomontage showing fluorescence after intracerebroventricular injection of Evans blue dye (grey). H: Low magnification photomontage of the same section showing occludin (green) and MECA 32 (red) immunoreactivities. I–K: High magnification images captured from the same section showing the honeycomb pattern of expression for occludin in cuboidal ependymal cells (green, empty arrow) (I) and tanycytes of the median eminence (green, empty arrow) (K) and the unorganized pattern of expression for occludin in tanycytes lining the arcuate nucleus (green, empty arrowhead) (J). L: Color-combined image of G and H. When injected into the cerebrospinal fluid, Evans blue diffusion is restricted to the arcuate nucleus and is found to diffuse neither into other hypothalamic nuclei nor into the median eminence. The honeycomb pattern of occludin expression corresponds to the non-diffusion of the dye, whereas the unorganized pattern of expression for occludin may be associated with diffusion of the dye into the arcuate nucleus. Sections are counterstained using Hoechst (blue) to visualize cell nuclei and recognize the morphological limits of each hypothalamic structure. 3V, third ventricle; ARH, arcuate nucleus of the hypothalamus. Scale bar = 20μm in E and K; 100μm in F and L.