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glia22535-sup-0001-suppFig1.tif14979KSupplemental Figure 1: Details of the myelin spot assay. Myelin (1µl) was placed on a coverslip in 3–4 spot roughly equidistant from one another (A). Markers of immature (O4; red) or mature oligodendrocytes, but not OPC markers (NG2; green) diffusely label the myelin spot. These markers were used to determine if the cells were located “On Myelin” or “Off Myelin” (B). Due to the granular appearance of myelin, it was possible to distinguish it from cellular structures. Thus, myelin was not a source of false positives during the quantification of cellular markers. Nuclei are stained with Hoechst (blue).
glia22535-sup-0002-suppFig2.tif13284KSupplemental Figure 2: Typical purity of oligodendrocyte culture. OPCs were expanded in mitogens for 1 week, before being plated onto coverslips and fed with media lacking mitogens to induce differentiation. After 2 days in vitro, the majority of OPCs differentiated and express the immature oligodendrocyte marker, O4 (red; A). A small proportion of cells are maintained as OPCs as indicated by the marker NG2 (green; B). NG2 expression and O4 expression are typically mutually exclusive (example with white arrow). Those cells expressing both OPC markers (NG2 or PDGFR-α) and O4, were considered to be O4-positive. There was a small percentage of contaminating astrocytes (GFAP; green) (C). Here, 66% of cells label with O4, 23% of cells label with NG2 and 2% of cells label with GFAP, indicating that the vast majority of cells (89%) express oligodendrocyte lineage markers (D). We did not find evidence of neuronal (βIII-tubulin) or microglial (IBA1) contamination. Nuclei are stained with Hoechst (blue). Error bars are standard error of the mean (D).

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