Supplementary Figure 9: Lineage tracing of microglia source during repopulation following demyelination.

a. Flow cytometry gating on CD11b⁺CD45^lo microglia, isolated from in vivo demyelinated lesions in Nes-CreERT2;RCL-tdT mice. This experiment was performed on 3 mice per time point. b. Mean number of tdT(Nestin)+ CD11b⁺CD45^lo cells ± s.e.m. at 3, 7, and 10 days post lysolecithin (dpl) detected in in vivo lesions. N = 3 mice per time point. c. Schematic of 4-hydroxytamoxifen (4-OHT) treatment in Nes-CreERT2;RCL-tdT explants ex vivo. d. Representative explant from Nes-CreERT2; RCL-tdT mice at 21 days in vitro (DIV) with 4-OHT treatment immunostained with Nestin (green) shows high recombination efficiency. Scale bar, 20 μM. This experiment was performed with 3 mice. e. Explants from Nes-CreERT2;RCL-tdT mice at 0 dpl, 2 dpl, and 7 dpl immunostained for IBA-1 (green). Scale bar, 20 µm. e’: magnified view of tdT+ cells in 0 dpl and 2dpl, showing an area of high tdT+ IBA-1+ cell density at 2 dpl. Scale bar, 25 µm. Arrows demonstrate tdT+ IBA-1+ cells. This experiment was performed with 4 mice per time point. f. Mean proportion of IBA-1+ cells positive and negative for tdTomato(Nestin) at 0-2 dpl ± s.e.m in explants. N = 4 mice per time point. **P = 0.00154 0 dpl vs 2 dpl (Kruskal-Wallis test, Dunn’s Multiple Comparison post-test). g. Explants at 21 DIV show that tdT(Nestin)+ cells are positive for Musashi-1 (Msh-1; green) and Sox-2 (green), but negative for GFAP (green). Scale bar, 20 µm. This experiment was performed with 3 explants. h. Schematic of 4-OHT treatment in Cx3cr1-CreER;RCL-tdT explants. i. Representative explant from Cx3cr1-CreER; RCL-tdT mice at 21 days in vitro (DIV) with 4-OHT treatment immunostained with IBA-1 (green) shows partial recombination. Scale bar, 20 μm. j. Representative explant from Cx3cr1-CreER; RCL-tdT stained for oligodendrocyte lineage cells (Olig2+; green) shows no co-localization with tdTomato (red). Scale bar, 50 μM. This experiment was performed with 3 explants. k. Explants from Cx3cr1-CreER;RCL-tdT mice at 0 dpl and 2 dpl immunostained for IBA-1 (green) and Nestin (magenta). Scale bar, 20 µm. This experiment was performed with 4 mice per time point. l. Mean proportion of IBA-1+ cells positive and negative for tdTomato(Cx3cr1) at 0-2 dpl ± s.e.m in explants. N = 4 mice per time point. m. Mean number of IBA-1+ cells positive for tdTomato(Cx3cr1) at 0-3 dpl ± s.e.m. in explants. N = 4 mice per time point. n. Mean proportion of tdTomato(Cx3cr1)+ cells positive and negative for Nestin at 0-2 dpl ± s.e.m in explants. *P = 0.0106, 0 dpl vs 2 dpl (Kruskal-Wallis test, Dunn’s Multiple Comparison post-test). N = 4 mice per time point. o. Mean number of tdTomato(Cx3cr1)+ cells positive for Nestin at 0-3 dpl ± s.e.m in explants. N = 4 mice per time point.