Panel A shows fluorescence microscopy images of Iba1, ChAT, GFP, and CD47 mRNA in WT spinal cord at P5 and P10. Panel B shows fluorescence microscopy images comparing NeuN, ChAT, Iba1, and CD47 mRNA in WT and CD47 deficient spinal cords. Panel C shows fluorescence microscopy images of parvalbumin and CD47 mRNA expression in WT dorsal root ganglia at P5. Panel D shows fluorescence microscopy images of parvalbumin and SIRPα mRNA expression in WT dorsal root ganglia at P0. Panel E shows fluorescence microscopy images of ChAT, Iba1, and SIRPα mRNA in the WT ventral horn at P0. Panel F shows fluorescence microscopy images of Iba1, CD47, and SIRPα mRNA in the WT ventral horn at P0.
Neurons express CD47, while microglia express SRIPα. (A) Z-stack confocal images (total distance in Z-axis: 3.5 μm) from a P5 L4 spinal cord showing motor neurons (ChAT; blue), microglia (Iba1; red), and CD47-mRNA (RNAscope, in green) at P5 (top row). At P10 (bottom row), images show motor neurons (ChAT; blue), astrocytes (GFAP, red), and CD47-mRNA (green). Scale bar in a1 and a4: 10 μm. Middle and right columns correspond to higher magnification images marked by dotted boxes in a1 and a4, respectively. White arrows indicate the CD47-mRNA puncta. Scale bar in a2, a3, a5, and a6: 5 μm. (B) Confocal images of CD47-mRNA puncta in interneurons, labelled by NeuN (blue), motor neurons (ChAT; red), and CD47-mRNA (green). Scale bar in b1: 50 μm. b2' image shows NeuN signal from dotted box in b1. b2'' shows CD47 mRNA signal for the same area as b2'. Dotted lines mark the perimeter of the neuronal soma. Bottom row of images shows validation of CD47-mRNA specificity using an RNAscope assay in a CD47−/− mouse, with immunohistochemistry against ChAT (b4) and Iba1 (b5). Scale bar in b3, b4, b5: 10 μm. (C) Confocal images of dorsal root ganglia (DRG) showing immunoreactivity against PV (blue), DAPI signal (white), and RNAscope probe against CD47-mRNA (green). Scale bar: 50 μm. (D) Confocal image of PV (blue), DAPI (white), and SIRPa-mRNA (red). Scale bar: 50 μm. (E) Confocal image showing motor neurons (ChAT, blue), microglia (Iba1, green), and SIRPα-mRNA (red). Scale bar: 50 μm. Insets on the right, show higher magnification of Iba1 and SIRPα-mRNA in dotted box area. (F) Single optical plane of confocal image showing immunoreactivity for microglia (Iba1, blue) and CD47 (red) and RNAscope signal against SIRPα-mRNA (green). Scale bar: 5 μm.