Fig 2: Percentage of Iba‐1+ cells expressing TMEM119 or P2RY12, in the brain tissue of controls and stroke patients and in different stages of ischemic infarct lesions. There is a significant reduction of the percentage of Iba‐1+ cells expressing these markers in the normal brain tissue of stroke and this percentage is further reduced within the lesions.

Fig 3: Density of homeostatic microglia (TMEM119 + , P2RY12 +) in temporal and occipital white matter (WM) was significantly decreased in demented but not in resilient when compared to control brains (a). Representative photomicrographs of TMEM119 + and P2RY12 + microglia on sections from control, resilient and demented cases containing temporal WM are displayed on the right (b). C Control (Braak 0-II); R Resilient (Braak III/IV); D Demented (Braak III/IV); *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Scale bar 50 μm

Fig 4: TSPO expression in microglial phenotypes and lymphocytes. Resident microglia expressing TSPO in an active and chronic active lesion and the periplaque white matter (A and B) as well as in NAWM (C). Resident microglia did not show any significant difference in TSPO expression compared to control or NAWM (D). Overview of expression of the homeostatic marker P2RY12 with TSPO in an active and chronic active lesion and the periplaque white matter (E and F), as well as in NAWM (G). A loss in homeostatic microglia expressing TSPO was found in active and chronic active lesions stages (H). Both TSPO+ and TSPO− microglia were found expressing TMEM119 or P2RY12 in multiple sclerosis lesions (black arrowheads; insets; C and G). In contrast, active and chronic active lesions showed an increase in CD206+CD40+ cells expressing TSPO (I and J). T cells (CD3) showed low expression of TSPO in multiple sclerosis lesions (K) in contrast to B cells (CD20) which showed strong localization with TSPO (L). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Scale bars in A, B, E and F = 200 μm; C, G, K and L = 50 μm, I = 25 μm. Insets are digitally zoomed to ×800. CA = chronic active.

Fig 5: Co-expression of microglia/macrophage markers in multiple sclerosis lesions. (A–J) Double staining for P2RY12 (green) and TMEM119 (red) shows that in the normal white matter of controls most microglia co-express P2RY12, although there are some, which are single positive for TMEM119 (A). In the normal-appearing white matter of multiple sclerosis a similar pattern as in control normal white matter is seen, but some of the TMEM119 single positive cells have a macrophage-like phenotype (B and insetC). P2RY12 is massively reduced in TMEM119+ cells in initial lesions (D and insetE) and there is a further reduction of P2RY12 expression in early active lesions, which still contain high numbers of TMEM119+ cells (F) and in the active centre of active lesions, which contain only few TMEM119+ cells (G). In inactive lesions, only few microglia-like cells are seen, but the majority of these cells co-express P2RY12 and TMEM119 (H). The insetsI and J show the expression of both antigens in a single double stained cell. (K–S) In the normal white matter of controls, the majority of microglia identified by the expression of P2RY12 (K) or TMEM119 (L) were activated and also expressed the phagocytosis-associated marker CD68. (M) An example of the normal-appearing white matter of an multiple sclerosis patient with numerous TMEM119+ microglia cells, while CD163 expression is restricted to perivascular macrophages. In N, activated microglia in initial lesion areas of a pattern III lesion are shown, which co-express TMEM119 and CD68. The edge of an active pattern III lesion is shown in O. In the initial lesion areas, most microglia cells express p22phox (NADPH oxidase) but are negative for CD163, although some microglia co-express both antigens (inset). In the early active lesion edge, there is still a dominant expression of p22phox, while co-expression of p22phox with CD163 increases towards the more advanced lesion parts. A similar profile is shown in P, which documents that CD206 appears in p22phox-positive macrophages predominately in advanced lesion stages. (Q–S) The expression profile of the markers in macrophages in the inactive lesion centre of active plaques. Some of the CD68+ (Q) or CD206+ macrophages (R) are co-labelled with TMEM119. In addition, some pro-inflammatory activated macrophages positive for p22phox co-express the anti-inflammatory M2 marker CD206 (S). Scale bars = 100 µm.