(A) Morphological analysis of microglia shows an increase in the circularity index, indicating a more activated phenotype, in SCFA- compared to control-treated germ-free (GF) APPPS1 mice. Iba1 (red) has been used as microglial marker. For each group, each shade of color represents the microglia from a single mouse (U test; n(Ctrl) = 6, n(SCFA) = 5). (B) smFISH analysis of microglial cells (red, Cx3cr1 mRNA particles) surrounding amyloid plaques (green, anti-Aβ clone 2D8) shows an increased number of Cx3cr1-positive cells (>4 puncta) clustering around Aβ plaques in SCFA- compared to control-supplemented GF APPPS1 mice. DAPI (blue) was used as nuclear dye (U test; n(Ctrl) = 5, n(SCFA) = 5 mice, 5 FOV per mouse). (C) Experimental outline for APPPS1 brain homogenate injection into a WT mouse brain showing the injection site (red dot). (D) smFISH analysis of Cx3cr1-positive cells (>4 puncta) surrounding the APPPS1 brain homogenate injection site (analysis area relative to injection site for all brains) showing an enhanced recruitment of microglial cells in specific pathogen-free (SPF) versus GF WT mice. (E) APPPS1 brain homogenate injection induces higher microglial activation in SPF in comparison to GF WT mice as shown by the higher amount of Trem2 mRNA puncta (red) per Cx3cr1-positive (green) microglia. DAPI (blue) was used as nuclear dye. In D and E, each different shade of color represents the microglia from a single mouse (U test, n(GF) = 3, n(SPF) = 3; two individual experiments, 120 analyzed images (with multiple microglia) per mouse). (F) Microglial recruitment and Aβ plaque uptake were histologically quantified in SCFA- and control-supplemented SPF APPPS1 mice. Although significantly more CD68-positive (green) and Iba1-positive (red) microglial cells were located at Aβ plaques (white, anti-Aβ clone 3552), the number of Aβ-positive microglia was significantly reduced in the SCFA- compared to the control-treated group. Values are expressed as percentages and normalized to the control-treated group (unpaired T-test; n(Ctrl) = 4, n(SCFA) = 5; one individual experiment).