Supplementary Figure 2: Increase in myelin–microglia contact, lysosomal number and size in the white matter of the brain with age

(a) The number of Iba1-positive microglia was quantified in the corpus callosum of 2, 9, 18 and 24 months old mice (n=4 mice per group, mean +/- s.d., Kruskal-Wallis-Test, *P=0.016, Chi-Quadrat=10.385, df=3, followed by Mann Whitney post-hoc test indicates significant difference in 9 vs 24 months, ^*P=0.0411, in 18 vs 24 months, *P=0.044). Each dot represents the mean value of 3 brain slices per mouse (b) Confocal image of microglia (Iba1, red) and myelin sheaths (MBP, green) in the striatum of 18 month old mice showing the contact of microglia with myelin connected to a sheath. Quantification of microglia cell area in contact with myelin (n=4 mice per group, mean +/- s.d., one-way ANOVA, ***P<0.0001, F=20.24, df=39, followed by Bonferroni’s post hoc test indicates significant difference in 4 vs 9, ***P=0.0001, t=5.334 and in 4 vs 18 months, ***P<0.0001, t=5.968). 30 cells per group of animals were analyzed. Scale bars: 7 µm (overview), 2 µm (Zoom in), 1 µm (clipped 3D). (c) Visualization and quantification of Lamp1 (red) positive microglia (Iba1, green) in wild type mice. Quantification shows % of Lamp1-positive microglia (n=3-4 mice per group, mean +/- SD, one-way ANOVA, ***P=0.0006, F=15.75, df=12). Quantification shows the size of Lamp1-positive lysosomes in microglia in the white and grey matter (n=4 mice per group, mean +/- s.d., two-way ANOVA, brain region effect: ***P<0.0001, F=39.04, followed by Bonferroni’s post hoc test, ***P=0.0006, t=5.180, *P=0.0213, t=3.505). Each dot represents the mean value of 3 brain slices per mouse Scale bars: 20µm. (d) Galectin3/Mac2 staining of brain section of 24-month-old wild type mice. Zoom in show corpus callosum (a), anterior commissure (b) and medial septal nucleus (c) Scale bars: 500µm (overview); 50µm (Zoom in). All images are are representative of three independent experiments.