Fig. 3

Csf1r^+/− microglia show an inflammatory and phagocytic phenotype in vivo and in vitro. A Representative Iba1 (green) and CD68 (red) immunofluorescent images of Csf1r^+/+ and Csf1r^+/− primary microglia. Note the amoeboid-like morphology of Csf1r^+/− microglia. B Statistical analysis of soma size in Csf1r^+/+ and Csf1r^+/− microglia (n = 3 independent experiments). C Statistical analysis of the percentage of Iba1 (green) and CD68 (red) double-positive (Iba1⁺CD68⁺) microglia relative to Iba1⁺ microglia per field (n = 3 independent experiments). D Representative Iba1 (green) and CD68 (red) immunofluorescent images of brain slices from Csf1r^+/+ or Csf1r^+/− mice at the age of 10 to 14 months. Three-dimensional (3D) reconstructions of representative Iba1⁺CD68⁺ microglial cells in the white box. E Statistical analysis of process number in Iba1⁺ microglia in the hippocampus of 10- to 14-month-old Csf1r^+/+ (n = 5 mice, 61 cells) and Csf1r^+/− (n = 6 mice, 107 cells) mice. F Statistical analysis of the percentage of Iba1⁺CD68⁺ microglial cells relative to the number of Iba1⁺ microglia in hippocampus samples of 10- to 14-month-old Csf1r^+/+ (n = 5 mice) and Csf1r^+/− (n = 6 mice) mice. G TNF-α and IL-1β protein levels in forebrain samples of Csf1r^+/+ or Csf1r^+/− mice (n = 5 mice). H TNF-α and IL-1β protein levels in Csf1r^+/+ or Csf1r^+/− microglia (n = 3 independent experiments). I Representative images of Iba1⁺ microglia after phagocytosis of red fluorescent dye (pHrodo)-labeled myelin sheaths. J Quantification of the ratio of Iba1⁺ pHrodo myelin-engulfing (pHrodo⁺) microglia to total Iba1⁺ microglia (n = 3 independent experiments). K The phagocytic ability of microglia on myelin sheaths quantified by mean pHrodo fluorescent intensity in Iba1⁺ cells (n = 3 independent experiments). Data are presented as means ± SEM. P-values were calculated using unpaired two-tailed Student’s t-test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001