β-dystroglycan or laminin immunostaining combined with immunostaining against GFAP or vimentin. Double labeling is performed on frontal sections. Asterisks indicate vessel-containing in-foldings of the pial surface surrounded by glial processes. (A) GFAP (red) and laminin (green) double immunolabeling. GFAP immunoreactivity marks a ‘shell’ that is free from laminin-immunopositive vessels. Arrows indicate glial processes around the laminin-immunolabeled vessels in the ‘core’. Double arrows mark glial processes extending to the pial surface. Inset: enlarged detail of the pial in-folding near the asterisk. (B) Vimentin (red) and laminin (green) double immunolabeling. Arrowheads point to flat ependyma covering the central part of the subfornical organ (SFO). Double arrowheads mark cuboidal ependyma covering the lateral part of the SFO. Arrows are as in (A). Half arrows mark non-ependymal long processes. (C) Vimentin (green) and β-dystroglycan (red) double immunolabeling. Arrows show that every β-dystroglycan-immunolabeled vessel is surrounded by glial processes. (D) GFAP (green) and β-dystroglycan (red) double immunolabeling. Arrows are as in (C); double arrows mark glial processes extending to the pial surface. (E and F) Vimentin-immunoreactive (red) glial processes around laminin-immunoreactive (green) vessels. (E) is an enlargement of (B). Half arrows mark the continuous perivascular glia, non-ependymal long processes. Arrowhead shows continuous perivascular glia, ependymal process. Bent arrows show discontinous perivascular glia with club-like endings. (G) RECA-1 (red) and laminin (green) double immunolabeling. Arrowheads show laminin-immunoreactive vessels. Double arrowheads show vessels with minimal or no laminin immunoreactivity. (H) Subsequent immunostaining for GFAP (green) on the vessels seen in (G). Arrows show GFAP-immunoreactive glial processes; other marks are as in (G). Scale (A–D) 100 µm (inset, 50 µm); (E–H) 25 µm. Abbreviations: 3V, third ventricle; VCH, ventral hippocampal commissure.