Fig. 5

Altered expression of activation markers in hippocampal microglia from GF 5xFAD mice. Expression levels (counts per million) of (a) ApoE, (b) P2ry12, (c) Clec7a and (d) Itgax in hippocampal microglia from SPF, GF and ABX-treated 5xFAD and age-matched WT mice, based on RNA-seq data depicted in Fig. 4. Each symbol represents one mouse. Data are presented as mean ± s.e.m. (e) Representative immunofluorescence images of Iba1⁺ (red), P2ry12⁺ (green) microglia and TR⁺ (white) Aβ on parasagittal hippocampal sections from SPF, GF and ABX-treated 5xFAD mice. Nuclei were stained with DAPI (blue). Scale bar: 50 μm. White arrowheads indicate P2ry12^dim/Iba1⁺ microglia and non-filled arrowheads show P2ry12^bright/Iba1⁺ microglia. Quantification of the percentage of (f) total parenchymal P2ry12^dim/Iba1⁺ microglia (g) TR⁺ plaque-associated P2ry12^dim/Iba1⁺ microglia and (h) non-plaque-associated P2ry12^dim/Iba1⁺ microglia in hippocampi of SPF, GF and ABX-treated 5xFAD mice. (i) Representative immunofluorescence images of Iba1⁺ (red) ApoE⁺ (green) microglia and TR⁺ (white) Aβ on parasagittal hippocampal sections from SPF, GF and ABX-treated 5xFAD mice. Nuclei were stained with DAPI (blue). Scale bar: 50 μm. White arrowheads indicate ApoE⁺/Iba1⁺ microglia and non-filled arrowheads show ApoE⁻/Iba1⁺ microglia. Quantification of the percentage of (j) total parenchymal ApoE⁺/Iba1⁺ microglia (k) TR⁺ plaque-associated ApoE⁺/Iba1⁺ microglia and (l) non-plaque-associated ApoE⁺/Iba1⁺ microglia in hippocampus from SPF, GF and ABX-treated 5xFAD mice. (m) Representative immunofluorescence images of Iba1⁺ (red) Clec7a⁺ microglia (green) and TR⁺ (white) on coronal hippocampal sections from SPF, GF and ABX-treated 5xFAD mice. Nuclei were stained with DAPI (blue). Scale bar: 50 μm. White arrowheads indicate Clec7a⁺/Iba1⁺ microglia and non-filled arrowheads show Clec7a⁻/Iba1⁺ microglia. Quantification of the percentage of (n) total parenchymal Clec7a⁺/Iba1⁺ microglia (o) TR⁺ plaque-associated Clec7a⁺/Iba1⁺ microglia and (p) non-plaque-associated Clec7a⁺/Iba1⁺ microglia in hippocampus of SPF, GF and ABX-treated 5xFAD mice. Each symbol represents one mouse. At least three slides were examined per individual mouse. Data are presented as mean ± s.e.m. Significant differences were determined by one-way ANOVA (*P < 0.05, **P < 0.01, ***P<0.001). Data are representative of two independent experiments. (q) Representative cytometric graph of CD11c⁺ labelled microglia from SPF (black line), GF (red line) and ABX-treated (blue line) 5xFAD mice and respective age-matched WT mice (dashed lines), compared to the isotype control (green line). In addition, quantifications of (r) percentages and (s) geometric mean fluorescence intensities (gMFI) of CD11c⁺ microglia cells are depicted. Each symbol represents one mouse. Data are presented as mean ± s.e.m. Significant differences were determined by two-way ANOVA followed by Bonferroni’s post-hoc comparison test or by one-way ANOVA followed by Tukey’s post-hoc comparison test (*P<0.05, ***P < 0.001). Data are representative of three independent experiments