Journal: BMC Neurology

Article Title: NG2 and phosphacan are present in the astroglial scar after human traumatic spinal cord injury

doi: 10.1186/1471-2377-9-32

Figure Lengend Snippet: Normal distribution of NG2, neurocan, versican and phosphacan in the human spinal cord . Transverse sections of control human spinal cords. A: NG2 immunohistochemistry reveals small stellate-shaped cells distributed homogeneously in white matter regions of human spinal cord (arrows). B: In the white matter, neurocan immunoreactivity is observed in the wall of a small blood vessel (arrow). Furthermore, a reticular staining pattern can be seen. C: In a dorsal nerve root, neurocan staining is present in myelin sheaths. D: Versican immunoreactivity is scattered in a dorsal nerve root and can be found in myelin sheaths of small diameter axons. E: Phosphacan immunohistochemistry reveals a fine reticular staining pattern in the gray matter. F: In a dorsal nerve root, phosphacan-immunopositive myelin rings can be observed. ( A-F magnification × 320).

Article Snippet: The primary antibodies used were: monoclonal mouse anti-human NG2 (clone B5, undiluted cell culture supernatant from ATCC cultures and antibody 9.2.27, diluted 1:50; gift from Prof. R. Reisfeld, Scripps Research Institute), monoclonal mouse anti-neurocan (Chemicon, diluted 1:100), polyclonal rabbit anti-versican (Acris antibodies, diluted 1:1.000), monoclonal mouse anti-phosphacan (Chemicon, diluted 1:500), polyclonal rabbit anti-GFAP (DAKO, diluted 1:2.500), polyclonal rabbit anti-myelin basic protein (MBP) (Chemicon, diluted 1:1.000) and polyclonal rabbit anti-neurofilament (NF, Sigma-Aldrich, diluted 1:2.000).

Techniques: Immunohistochemistry, Staining

Journal: BMC Neurology

Article Title: NG2 and phosphacan are present in the astroglial scar after human traumatic spinal cord injury

doi: 10.1186/1471-2377-9-32

Figure Lengend Snippet: The cellular and molecular composition of the scar in human SCI after early and long survival times . Transverse sections of the human spinal cord of control cases and at early and late survival times after SCI. The schematic diagrams in the upper right corner indicate the region from where the actual picture was taken (black rectangle). A: Co-localisation of phosphacan (green) and MBP (red) confirms the presence of the CSPG in the myelin sheaths of control dorsal root axons. B: Double immunofluorescence with phosphacan (green) and laminin (red) reveals CSPG-immunopositive myelin rings surrounded by laminin-positive endoneurium in a dorsal nerve root of control spinal cord. C: Double immunofluorescence with NG2 (red) and CD68 (green) in macrophages at the lesion epicentre, 10 days after injury. D: One year after SCI, staining for versican (red) and NF (green) in sections from the lesion epicentre demonstrated individual and bundled regenerated nerve fibres that were surrounded by a versican-positive endoneurium in the ECM. E-G: In sections from the lesion epicentre of the same case, double immunofluorescence for NF (green) and neurocan ( E , red), versican ( F , red) or phosphacan ( G , red) revealed nerve fibres surrounded by a CSPG-positive endoneurium. H-I: In the intermediate zone of the same case, GFAP (green) and NG2 ( H , red) and phosphacan ( I , red) immunofluorescence demonstrated the close overlap of all three proteins in the astroglial scar after human SCI. J: In an adjacent section, NF (green) and phosphacan immunohistochemistry revealed occasional, small, nerve fibres still present within the CSPG-rich ECM of the astroglial scar. ( A-J magnification × 400).

Article Snippet: The primary antibodies used were: monoclonal mouse anti-human NG2 (clone B5, undiluted cell culture supernatant from ATCC cultures and antibody 9.2.27, diluted 1:50; gift from Prof. R. Reisfeld, Scripps Research Institute), monoclonal mouse anti-neurocan (Chemicon, diluted 1:100), polyclonal rabbit anti-versican (Acris antibodies, diluted 1:1.000), monoclonal mouse anti-phosphacan (Chemicon, diluted 1:500), polyclonal rabbit anti-GFAP (DAKO, diluted 1:2.500), polyclonal rabbit anti-myelin basic protein (MBP) (Chemicon, diluted 1:1.000) and polyclonal rabbit anti-neurofilament (NF, Sigma-Aldrich, diluted 1:2.000).

Techniques: Immunofluorescence, Staining, Immunohistochemistry

Journal: BMC Neurology

Article Title: NG2 and phosphacan are present in the astroglial scar after human traumatic spinal cord injury

doi: 10.1186/1471-2377-9-32

Figure Lengend Snippet: The cellular and molecular composition of the scar in human SCI after long survival times . Transverse sections of the lesioned human spinal cord at 1 year (A-C) and 20 years (D-I) after injury. The schematic diagrams in the upper right corner indicate the region from where the actual picture was taken (black rectangle). A: NG2 staining revealed a network of irregular lamellae in the ECM at the lesion epicentre. B-C: In near adjacent sections, versican ( B ) and phosphacan ( C ) immunohistochemistry demonstrated fibre-like structures either singly or in small bundles. D: Twenty years after SCI, nerve root-like structures at the lesion epicentre demonstrated neurocan-positive myelin rings. E: In a near adjacent section, phosphacan immunoreactivity was also present in myelin sheaths surrounding axons in neuromas. F: Versican immunopositive myelin rings surrounding regenerated nerve fibres were more scattered and of a small diameter in the nerve root-like structures at the lesion epicentre. G: Twenty years after SCI, diffuse but elevated levels of NG2 immunoreactivity were still associated with the ECM of the connective tissue scar (lower part of image) as well as with the astroglial scar of the intermediate zone (upper part of image). At the lesion epicentre, NG2 staining was located on more loosely arranged sheets of basal lamina-like ECM. In the astrocytic scar, NG2 was associated with a dense irregular network of processes. Arrows demarcate the border between the connective tissue component of the lesion and the adjacent astrocytic scar. H: In a near adjacent section, diffuse phosphacan immunoreactivity was also associated with the dense network of processes of the astroglial scar. No immunoreactivity was associated with the ECM of the connective tissue scar. I: GFAP staining strictly delineated the interface between the astroglial scar and the connective tissue scar. ( A-I magnification × 320).

Article Snippet: The primary antibodies used were: monoclonal mouse anti-human NG2 (clone B5, undiluted cell culture supernatant from ATCC cultures and antibody 9.2.27, diluted 1:50; gift from Prof. R. Reisfeld, Scripps Research Institute), monoclonal mouse anti-neurocan (Chemicon, diluted 1:100), polyclonal rabbit anti-versican (Acris antibodies, diluted 1:1.000), monoclonal mouse anti-phosphacan (Chemicon, diluted 1:500), polyclonal rabbit anti-GFAP (DAKO, diluted 1:2.500), polyclonal rabbit anti-myelin basic protein (MBP) (Chemicon, diluted 1:1.000) and polyclonal rabbit anti-neurofilament (NF, Sigma-Aldrich, diluted 1:2.000).

Techniques: Staining, Immunohistochemistry

Journal: Frontiers in Cellular Neuroscience

Article Title: Neurocan Inhibits Semaphorin 3F Induced Dendritic Spine Remodeling Through NrCAM in Cortical Neurons

doi: 10.3389/fncel.2018.00346

Figure Lengend Snippet: Localization of Neurocan in mouse medial frontal cortex (MFC) by immunogold labeling and electron microscopy. (A) Electron micrograph of MFC layer 2/3 at P18, showing immunogold labeling of Neurocan near the plasma membrane adjacent to a spine (Sp) and axon terminal (AT; arrows). (B) Neurocan labeling in the extracellular space near an axon terminal (AT; arrow) at P18 [Nucleus (Nuc) and cytoplasm (Cyto)]. (C) Accumulation of Neurocan (arrows) in extracellular space and along the plasma membrane of a dendrite (D) at P18. Mitochondria (M) were unlabeled. (D) Neurocan labeling adjacent to axon terminals (AT) at P18. (E) Neurocan labeling at neck of spine (Sp) and near excitatory synapses (arrows) at P80. Scale bar = 1 μm. (F) Validation of Neurocan antibody specificity by immunoperoxidase staining of COS-7 cells transfected with Neurocan-AP or AP alone in the APtag5 vector, using Neurocan antibodies or no primary antibody. An antibody dilution series was carried out in pilot experiments. Scale bar = 50 μm.

Article Snippet: Samples were blocked in PBS, 10% donkey serum, 0.3% Triton X100, then incubated with Neurocan antibodies (1:500, R&D) for 24 h at 4°C, then with anti-sheep Alexa Fluor 488 secondary antibody (1:500).

Techniques: Labeling, Electron Microscopy, Immunoperoxidase Staining, Transfection, Plasmid Preparation

Journal: Frontiers in Cellular Neuroscience

Article Title: Neurocan Inhibits Semaphorin 3F Induced Dendritic Spine Remodeling Through NrCAM in Cortical Neurons

doi: 10.3389/fncel.2018.00346

Figure Lengend Snippet: Cell binding and Neurocan interaction with NrCAM. (A) COS-7 cells transfected with vector alone (pCAGGS-IRES-mEGFP) or pCAGGS-NrCAM-IRES-mEGFP were pre-treated with 8 nM Neurocan, then fixed and subjected to immunofluorescence staining without permeabilization to detect surface-bound Neurocan (red). Scale bar = 100 μm. (B) Mean fluorescence intensity (±SEM) of Neurocan immunofluorescence staining on the surface of COS-7 cells, as shown in panel A . NrCAM-expressing cells treated with Neurocan showed significantly greater levels of bound Neurocan than untreated cells. Fluorescence intensity in cells with vector alone treated with Fc or Sema3F-Fc was non-significant (ns). ∗ p > 0.05, t -test, n = 5 images each condition. (C) Lysates (50 μg) of cells transfected with vector alone or pCAGGS-NrCAM-IRES-mEGFP were treated with Neurocan as in panel A , and immunoblotted (IB) with Neurocan antibodies. Blots were reprobed with antibodies directed against GAPDH (loading control) or NrCAM (expression control). Representative immunoblots of three experiments are shown. (D) Mouse cortical neuron cultures from NrCAM null mice were transfected with vector alone or pCAGGS-NrCAM-IRES-EGFP, and pre-treated with 20 nM Neurocan before fixation and immunostaining to detect surface-bound Neurocan. In merged images of EGFP (green) and Neurocan (red), more Neurocan immunofluorescence was observed on the surface of neurons expressing NrCAM than on NrCAM null neurons with vector alone. Scale bar = 50 μm. (E) Mean fluorescence intensity (±SEM) of surface-bound Neurocan immunostaining on neurons in panel D NrCAM-expressing cells treated with Neurocan showed significantly greater levels of bound Neurocan than NrCAM-minus neurons. ∗ p > 0.05, t -test, and n = 10 neurons per condition. (F) ELISA of Neurocan-AP or control AP protein binding to NrCAM-Fc or positive control NCAM-Fc on protein A-coated microtiter wells. AP binding was detected colorimetrically with p-nitrophenylphosphate. The mean (±SEM) optical densities (OD 405) of Neurocan-AP bound to NrCAM-Fc or NCAM-Fc were significantly greater than control AP ( t -test and ∗ p > 0.05). (G) Recombinant human Neurocan was incubated in Tris buffered saline with purified Fc, Sema3F-Fc, or Sema3A-Fc proteins, then complexes were pulled down with Protein A/G Sepharose beads. Immunoblotting for Neurocan showed no binding of Neurocan to Fc or Sema3F-Fc, whereas Neurocan bound effectively to Sema3A-Fc. Blots were reprobed with anti-Fc antibodies to demonstrate that equivalent amounts of Fc fusion proteins were pulled down. Recombinant Neurocan (left lane) ran as a broad band between 250 and 130 kDa.

Article Snippet: Samples were blocked in PBS, 10% donkey serum, 0.3% Triton X100, then incubated with Neurocan antibodies (1:500, R&D) for 24 h at 4°C, then with anti-sheep Alexa Fluor 488 secondary antibody (1:500).

Techniques: Binding Assay, Transfection, Plasmid Preparation, Immunofluorescence, Staining, Fluorescence, Expressing, Western Blot, Immunostaining, Enzyme-linked Immunosorbent Assay, Protein Binding, Positive Control, Recombinant, Incubation, Purification