CCL2 expression in spinal cord of WT mice during EAE. (a–b) z-stack confocal images from spinal cord cryosections of WT mice at d16 EAE are shown, revealing staining of CCL2 (red), and CD31 or GFAP (green) to delineate the endothelial cells and astrocytes, respectively. CCL2 staining was isosurface rendered for enhanced spatial perspective. (a) WT mice express CCL2 both along the CD31+ microvascular endothelium, where staining appears aligned along the endothelial junctions, and within the perivascular space (left). (b) CCL2 staining is also associated with GFAP+ astrocytes (right). Insets show co-localization of CCL2 with CD31 or GFAP (yellow) in a single z-slice from the respective regions marked by the hatched white boxes, or CCL2 (red) channel alone. (c–f) Colloidal gold immuno-EM localization of CCL2 localization along microvessels in sections of spinal cord from mice at d16 EAE. (c) CCL2 immunoreactivity is localized within the inter-endothelial junction (arrow) and scattered throughout the endothelial cytoplasm. (d) A cluster of CCL2 immunoreactivity (arrow) is shown in close apposition to an endothelial vesicular structure that is near the plasma membrane. (e) Low magnification showing cross-section of a microvessel (possibly a postcapillary venule or small venule) and punctate distribution of CCL2 immunoreactivity in the perivascular space (arrows). (f) Higher magnification, revealing a high density of CCL2 immunoreactivity in and around what may represent astrocyte endfeet (arrows). Results are representative of 5–7 sections sampled from three mice in each group and two independent experiments. Scale bars are noted on the respective images.