Fig. 6

Cluster 13 may represent a stroke-induced subpopulation of microglia with increased CD45 expression and macrophage properties. A Feature plots showing two top highly expressed genes in cluster 13. B Representative images of Arg1 fluorescence in situ hybridization (FISH) in peri-infarct areas of Tmem119-Ai6 mouse brains on day 3 after permanent stroke. Arrows (white) indicate ZsGreen⁺Arg1⁺ microglia. C, D ZsGreen⁺ microglia in the stroke brain. Tmem119-Ai6 mice were subjected to permanent stroke. On day 3 after stroke, single-cell suspensions were prepared from contralateral (contra) and ipsilateral (ipsila) hemispheres for flow cytometric analysis. C CD45 expression was increased in a subpopulation of ZsGreen⁺ cells. Representative gated plots were shown for analysis of ZsGreen⁺ cells. D CD45^HiZsGreen⁺ cells likely correspond to cluster 13 microglia. ZsGreen⁺ cells in the CD45^HiCD11b⁺ population express CCR2, a signature marker of cluster 13. E, F ZsGreen^DimtdTomato⁺ cells in the stroke brain. Chimera mice (microglia labeled with ZsGreen and periphery immune cells labeled with tdTomato) were subjected to permanent stroke. On day 3 after stroke, flow cytometry analysis (E) and confocal imaging (F) were performed. E CD45⁺CD11b⁺ cells were further analyzed. ZG: ZsGreen, tdT: tdTomato. F Representative orthogonal views of confocal images show phagocytosis of ZsGreen⁺ microglia and tdTomato⁺ immune cells. The xy images are presented as maximal projections of z stacks. Arrows depict tdTomato fluorescent dots and arrow heads depict punctate structures with ZsGreen signal. Data are representative of at least three independent experiments