Extended data figure 6. Immunohistochemistry of specific CSPGs.

(a) Neurocan (NCAN) production by scar-forming astrocytes and non-astrocyte cells. Images show individual fluorescence channels of NCAN and GFAP immunohistochemistry from horizontal sections two weeks after severe SCI, in a representative WT mouse. Box denotes area of lesion core (LC) and astrocyte scar (AS) shown at higher magnification. Note that NCAN is produced both by GFAP-positive scar-forming astrocytes and by non-astrocyte cells (arrows) in the lesion core (LC). (b) NG2 (CSPG4) production by newly proliferated scar-forming astrocytes. Images show individual channels and various combinations of immunofluorescence staining for NG2, GFAP, tdT, BrdU (proliferation marker) and DAPI showing astrocytes in a mature SCI scar. The images are representative of findings from tdT-reporter mice⁵² injected with AAV2/5-GfaABC1D-Cre vector⁵⁴ into multiple sites of the uninjured spinal cord to label mature astrocytes. Three weeks after AAV2/5-GfaABC1D-Cre injection, the mice received a severe SCI and were administered BrdU from days 2–7 after SCI. The mice were perfused after two weeks after SCI. Images comparing individual fluorescence channels show that astrocytes labeled 1 and 3: (i) incorporated BrdU and thus are newly proliferated after SCI, (ii) express tdT reporter, (iii) express GFAP, the prototypical marker of reactive and scar-forming astrocytes, and (iv) express NG2 both intracellularly and along their cell surfaces. In contrast, astrocyte number 2 is also BrdU-labeled and expresses both tdT and GFAP, but does not appear to express detectable levels of NG2. (c) CSPG5 (Neuroglycan C) production by scar-forming astrocytes. Images show individual channels and various combinations of immunofluorescence staining for CSPG5 or GFAP. Note that CSPG5 is present within and along the processes of GFAP-positive scar-forming astrocytes (arrows).