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Figure 7


Fig. 7. Myelination in oligodendrocyte-neuron co-cultures is perturbed by dystroglycan-blocking antibodies. (A) Rat dorsal root ganglion (DRG) neuron cultures, which contain laminin {alpha}2. Immunocytochemistry was used to detect laminin {alpha}2 (green) and nuclei (DAPI; blue). (B) Rat DRG neurons co-cultured with oligodendrocytes. Myelinating oligodendrocytes in rat DRG neuron co-cultures are found in association with laminin {alpha}2. Double immunocytochemistry was used to detect laminin {alpha}2 (green) and myelin basic protein (MBP; red). Nuclei were visualized using DAPI (blue). (C) Western blots to detect laminin proteins. Lysates obtained from oligodendrocytes (OL), astrocytes (A) or DRG neuron cultures were immunoblotted with a monoclonal antibody against the laminin {alpha}2 subunit (lm {alpha}2) or a polyclonal antibody against laminin-1 (lm-1). Note that laminin-1 antibodies detect the laminin {alpha}1, ß1 and {gamma}1 subunits. (D) Rat DRG neurons were seeded with oligodendrocyte progenitors in the presence of dystroglycan-blocking or control antibodies. At 2 weeks, differentiated oligodendrocytes were visualized using MBP immunofluorescence (green) in conjunction with neurofilament (NF) immunofluorescence (red) to visualize the underlying neurite network. (E) The average number of MBP-positive cells per field was scored in cultures treated with control and dystroglycan-blocking antibodies. No significant difference was observed (n=4, error bars represent s.d.). (F) The average percentage of myelinating MBP-positive oligodendrocytes (OL) was scored in dystroglycan-blocking- and control-antibody conditions. Dystroglycan-blocking antibodies significantly reduced the percentage of myelinating oligodendrocytes compared with control antibodies (n=4, **P<0.01, error bars represent s.d.). (G) The average number of myelinating segments per field was scored in cultures treated with control and dystroglycan-blocking antibodies. Dystroglycan-blocking antibodies significantly reduced the number of segments per field compared with control antibodies (n=4, **P<0.01, error bars represent s.d.). (H) Representative plots depicting the correlation between the myelinating oligodendrocyte (OL) to total oligodendrocyte (OL) ratio and neurite density (neurite area fraction, %). (I) Average slope of plots depicted in H (n=4, ***P<0.001, error bars represent s.d.). Scale bars: 100 µm.





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