Fig. 6.

Detection of Gal₁-mCherry immunofluorescence in spinal cord. (A) Low-power photomicrograph (10 × objective) and (B–D) confocal images (20 × objective) of adult GalR1-mCherry knock-in lumbar spinal cord showing high levels of immunofluorescence in the superficial dorsal horn laminae I–II, with lesser levels in the lateral spinal nucleus (LSN, labelled ‘L’) and around the central canal (white asterisk) in lamina X (A–C), whereas little immunofluorescence was detected in the ventral horn (D). (E–H) Higher magnified confocal images (40 × objective) of mid superficial dorsal horn (E); lateral superficial dorsal horn and LSN (labelled ‘L’; F); lamina III/IV border (G); and lamina X (H; central canal, white asterisk); with examples of local cell bodies boxed. (I–L, M) Higher magnification confocal images (40 × objective, zoom 6 ×) of local neurons (boxed in row above) show distinct somatic cell membrane localization in highly fluorescent cells of the superficial dorsal horn (I), lamina III/IV border (J) and lamina X (M; location arrowed in N, mainly different plane), whereas in less fluorescent neurons cell surface localization may (K, arrow; insert merged 3 z-sections) or may not be detected (K–L; L, merged 4 z-sections). Note the multiple Gal₁-mCherry immunofluorescent puncta within neuronal processes (J, arrows; additional images in Supplementary Fig. 5A–F), and insert of M shows three main neuronal projections (each double arrows; merged 16 z-sections, 1 μm intervals). (N–P) Confocal images of medial spinal cord including a lamina IV neuron sending a process laterally to another Gal₁-mCherry expressing neuron (boxed, N; 40 × objective); as shown at higher magnification to show the neuronal process (O; objective 40 ×, zoom 3 ×; merged 8 z-sections, 1 μm intervals), with insert images showing somatic cell membrane localization of each cell (right cell, merged 3 z-sections); and at further magnification Gal₁-mCherry puncta are seen in the terminal process of the lamina IV neuron (arrows, P; objective 40 ×, zoom 8 ×; merged 7 z-sections, 1 μm intervals), with insert images of details from successive z-sections of the terminal process. FD is funiculus dorsalis. Each confocal image is a single optical section from a z stack unless indicated, with (B–D) at same brightness intensity, as are (E–H and N). Scale bars: 150 μm for 20 × objective; 70 μm for 40 × objective; 40 μm for 40 × objective with zoom 3 ×; and 15 μm for 40 × objective with either zoom 6 × or 8 ×.