Fig. 3: Comparison between capillary-associated microglia and parenchymal microglia.

a Representative images from a CX3CR1^GFP/+ adult brain showing microglia (green) capillaries (magenta) and Sall1 transcripts (gray) with capillary-associated microglia (CAMs, arrows) parenchymal microglia (PCMs, arrowheads). b Quantification of microglial Sall1 expression in CAMs and PCMs. n = 10 CAM or PCM cells from each of three mice. c Representative in vivo two-photon projection images from a CX3CR1^GFP/+ adult brain showing microglia (green) that are either capillary- (magenta) associated (arrows) or parenchyma-situated (arrowheads). d–f Quantification of microglial primary process numbers (d, n = 5 CAM or PCM cells from each of five fields of view from each of three mice), microglial cell body sizes and (e, n = 5 CAM or PCM cells from each of five fields of view from each of five mice) microglial whole-cell area between CAMs and PCMs (f, n = 5 CAM or PCM cells from each of three to five fields of view from each of four mice). g Representative in vivo two-photon projection images from a time-lapse movie collected from a CX3CR1^GFP/+ mouse following a laser-induced injury. Microglial processes are directed towards the laser injury over time. h–j Quantification of basal microglial motility (h, n = 3–4 CAM or PCM cells from each of three mice) number of responding processes per cell (i, n = 3–4 CAM or PCM cells each of five mice) and the percent of responding cells following the laser-induced injury between CAMs and PCMs (j). k A representative schematic showing the longitudinal imaging scheme for visualizing microglia and capillaries. l–o Representative two-photon projection image from a CX3CR1^GFP/+ adult brain during longitudinal imaging showing various microglial (green)–capillary (magenta) dynamics including stable interactions, which persist over the imaging period (m), crawling interactions in which the position on the capillary changes, but remains on the capillary (n) and “hop on,” in which parenchymal microglia relocate to a proximal capillary (o). p Quantification of the various types of dynamic CAMs over a 4-week imaging period. n = 3–5 fields of view from each of four mice. Data are presented as mean values ± SEM. *p < 0.05, and n.s. not significant. Two-sided unpaired Student’s t test.