Fig. 2. Nptx2 regulates complement activity in vivo.

(A) Shown on the left are representative images of C1q immunostaining in WT and Nptx2^KO mice brains. Scale bar, 1000 μm. The right shows quantification of C1q immunofluorescence in WT and Nptx2^KO brains. P values were determined by unpaired t test. n = 9 for WT; n = 6 for Nptx2^KO. Norm., normalized. (B to D) C1q (B), C4 (C), and C4b (D) concentration in WT and Nptx2^KO cortices was measured by ELISA. P values were determined by unpaired t test. n = 8 for WT; n = 9 for Nptx2^KO. (E) Shown are representative confocal images of CD68 (green) and Iba1 (magenta) immunostaining in WT and Nptx2^KO cortical layers II to V. Scale bars, 30 μm and 2 μm (inset). (F) CD68 volume was quantified in WT and Nptx2^KO cortical layers II to V. P values were determined by unpaired t test. n = 5 for WT; n = 7 for Nptx2^KO. (G) Representative Western blots and quantification show CD68 abundance in hippocampi from WT and Nptx2^KO cortices. P values were determined by unpaired t test. n = 12 for WT; n = 12 for Nptx2^KO. (H) The number of Iba1⁺ microglia in WT and Nptx2^KO whole brains was quantified. P values were determined by unpaired t test. n = 3 for WT; n = 3 for Nptx2^KO. (I to K) Shown are representative confocal images and three-dimensional (3D) surface rendering of immunostained CD68 (green), Vglut1 (magenta), and Homer1 (blue) in WT and Nptx2^KO cortical layers II to V (I). Scale bars, 5 μm and 1 μm (inset). Homer1 (J) and Vglut1 (K) puncta inside CD68⁺ lysosomes were quantified. P values were determined by unpaired t test. n = 4 for WT; n = 5 for Nptx2^KO. (L) Representative confocal images of PSD95-GFP and immunostained parvalbumin are shown in samples from PSD95-GFP^f/f:PV-Cre WT cortical layers II to V. Scale bars, 10 μm and 4 μm (inset). (M) Shown are representative confocal images of immunostained C1q (magenta) and PSD95-GFP (green) in PSD95-GFP^f/f:PV-Cre:WT and Nptx2^KO hippocampus CA1 strata radiata. White circles and white arrowheads indicate C1q colocalized with PSD95-GFP. Scale bars, 2 μm and 0.5 μm (inset). (N) The percentage of C1q + PSD95-GFP puncta in WT and Nptx2^KO hippocampi was quantified. P values were determined by unpaired t test. n = 5 for WT; n = 6 for Nptx2^KO. (O) PSD95-GFP puncta density was quantified in PSD95-GFP^f/f:PV-Cre:WT and Nptx2^KO hippocampi. P values were determined by unpaired t test. n = 3 for WT; n = 3 for Nptx2^KO. (P) Shown on the left are representative confocal images and mask of immunostained PV (gray), Homer1 (blue), and Vglut1 (magenta) in WT and Nptx2^KO cortical layers II to V. The bar graph on the right shows excitatory synapse density around somas of PV⁺ cells. P values were determined by unpaired t test. n = 4 for WT; n = 5 for Nptx2^KO. Scale bars, 5 μm. (Q) Shown on the left are representative confocal images and mask of immunostained NeuN (gray), Homer1 (blue), and Vglut1 (magenta) in WT and Nptx2^KO cortical layers II to V. Scale bars, 5 μm. The bar graph on the right shows normalized number of excitatory synapses around NeuN⁺ soma. P values were determined by unpaired t test. n = 4 for WT; n = 5 for Nptx2^KO. Scale bars, 5 μm. Data are presented as means ± SEM. Each dot represents data from one mouse.