Fig. 3

Complement promotes microglial engulfment of excitatory synapse in mouse hippocampus after surgery. (A) Immunofluorescence detection of VGluT1 (green) & Homer1 (red) colocalized puncta in the hippocampal stratum radiatum from the control and surgery group. Scale bars: 10 μm. (B) Quantification of VGluT1 positive puncta (n = 4 mice per group). (C) Quantification of Homer1 positive puncta (n = 4 mice per group). (D) Quantification of VGluT1 & Homer1 colocalized puncta (n = 4 mice per group). (E-F) Immunofluorescence detection of VGluT1 (green) & C1q (red) colocalized puncta in the hippocampal stratum radiatum from the control and surgery group. (E) Representative images, scale bar: 20 μm and 5 μm. (F) Quantification of C1q and VGluT1 colocalized puncta (n = 4 mice per group). (G-H) Immunofluorescence detection of VGluT1 (green) & C3 (red) colocalized puncta in the hippocampal stratum radiatum from the control and surgery group. (G) Representative images, scale bar: 10 μm and 4 μm. (H) Quantification of C3 and VGluT1 colocalized puncta (n = 4 mice per group). (I-J) Triple immunofluorescence staining of IBA1 (green), CD68 (blue) and VGluT1 (red) in the hippocampal stratum radiatum from the control and surgery group. (I) Representative images and three-dimensional (3D) reconstruction. Scale bars: 10 μm and 3 μm. (J) Quantification of VGluT1 positive puncta within microglial lysosomes (n = 21–27 cells from 4 mice per group). (K-L) Effect of C3 treatment on the phagocytosis activity of microglia cells in vitro. (K) Representative immunofluorescence images showing the fluorescent beads (green) inside the primary microglial cells after C3 or PBS treatment. Scale bars: 100 μm. (L) Quantification of the fluorescent beads taken up by primary microglial cells from (K) (left: 6–7 fields from 3 batches per group; right: 35–36 cells from 3 batches per group). Data in bar graph are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns, not significant; by two-tailed t-test