Panel A shows fluorescence microscopy images comparing ChAT, VGlut1, and C3 localization around motor neurons. Panel B shows fluorescence microscopy images displaying C3 expression in motor neuron somatic regions. Panel C shows fluorescence microscopy images displaying C3 expression in motor neuron dendritic regions. Panel D shows fluorescence microscopy images comparing ChAT, VGlut1, and C3 localization in CD47 deficient motor neurons. Panel E shows fluorescence microscopy images displaying C3 expression around CD47 deficient motor neuron somata. Panel F shows fluorescence microscopy images displaying C3 expression around CD47 deficient motor neuron dendrites. Panel G shows bar graph comparing C3 expression around motor neuron somata between WT and CD47 deficient mice. Panel H shows bar graph comparing C3 expression around motor neuron dendrites between WT and CD47 deficient mice. Panel I shows bar graph comparing C3 expression between somatic and dendritic regions in WT mice. Panel J shows bar graph comparing C3 expression between somatic and dendritic regions in CD47 deficient mice.
C3 expression in WT and CD47 −/− spinal cords. (A) Single optical plane confocal images from L4 motor neurons at P5 in WT mice. Immunoreactivity for ChAT (blue), VGluT1 (red), and C3 (green) (scale bar: 50 μm). Insets (a1 and a2) depict examples from somatic and dendritic areas, respectively. a1′ and a2′ = C3 (green), a1″ and a2″ = VGluT1 (red), and a1‴ and a3‴ = merged with ChAT (scale bar: 10 μm). (B and C) Higher magnification of C3 protein expression around motor neuron somata and (C) around motor neuron dendrites (scale bar: 10 μm). Insets b1–b3 depict C3 expression surrounding motor neuron soma, while c1–c3 correspond to dendritic areas (scale bar: 1 μm). (D) Single optical plane confocal images from L4 motor neurons at P5 in CD47−/− mice. Immunoreactivity for ChAT (blue), VGluT1+ synapses (red), and C3 (green) (scale bar: 50 μm). Insets (d1 and d2) depict examples from somatic and dendritic areas, respectively. d1′ and d2′ = C3 (green), d1″ and d2″ = VGluT1 (red), and d1‴ and d2‴ = merged with ChAT (scale bar: 10 μm). (E and F) (E) Higher magnification of C3 protein expression around motor neuron somata and (F) around motor neuron dendrites (scale bar: 10 μm). Insets e1–e3 depict C3 expression surrounding motor neuron soma, while f1–f3 correspond to dendritic areas (scale bar: 1 μm). All images (A–F) were acquired under identical scanning conditions. (G–J) Quantification of optical intensity of C3 expression from areas of motor neuron somata, and dendrites in WT and CD47−/− mice. Data points in bar charts were from three images (area: 25 μm2) per animal (shown in B and E images for somata and C and F images for dendrites). Colored data points are averages from individual mice. (G–J) (G) Somata: WT versus CD47−/−; (H) dendrites: WT versus CD47−/−; (I) WT: somata versus dendrites; (J) CD47−/−: somata versus dendrite. Three images were analyzed per animal. WT, N = 3 mice; CD47−/−, N = 3 mice. Statistical comparison was performed between averages from individual mice. ns: no significance; *P < 0.05, **P < 0.01. Unpaired t test.