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. 2017 Jan 18;140(3):599–616. doi: 10.1093/brain/aww351

Figure 2.

Figure 2

AAV.MBP.Cx47myc leads to expression of Cx47 and gap junction formation in oligodendrocytes of Cx47 knockout mice. Double immunostaining for Cx47 (red) and either the myc tag (A–C) or the oligodendrocyte marker CC1 (D–F) (green) shows Cx47 expression and formation of gap junction (GJ) like plaques (open arrowheads) at the pericarya and proximal processes of oligodendrocytes in the corpus callosum and internal capsule co-localizing with the myc immunoreactivity (A and B) and restricted to CC1+ oligodendrocytes in full vector injected mice, while untreated Cx47 knockout mouse brain shows no Cx47 or myc immunoreactivity (C and F). Some endogenous green fluorescence is present diffusely in the cytoplasm of all Cx47 knockout oligodendrocytes (including the untreated mice) due to the transgenic expression. Double immunostaining for Cx47 or myc and other cell markers including neuronal NeuN (G), astrocytic GFAP (H), and microglia Iba1 (I) confirms that Cx47 is not expressed in any of these cell types and is restricted to oligodendrocytes. Scale bars = 10 μm. (J) Quantification of the percentage of Cx47 expressing oligodendrocytes in the corpus callosum (CC), internal capsule (IC), ventral pons (corticospinal tract area) and cervical spinal cord (SC) anteriolateral white matter of n = 5 mice shows high expression ratios in all areas. (K) Counts of Cx47 gap junction plaques per oligodendrocyte confirm the re-establishment of oligodendrocyte gap junctions at levels above those of wild-type mouse brain. (L) Immunoblot analysis of brain lysates from three AAV.MBP.Cx47myc injected mice shows the presence of a faint Cx47 band that is absent from non-injected Cx47 knockout mice and is present in a wild-type mouse sample run as positive control (a non-specific band below the specific one is marked with asterisk). Blotting with a myc antibody also shows immunoreactivity in injected mice, which is absent from non-injected knockout and from the wild-type mouse. GAPDH blot is used for loading control. (M) Quantification of band intensity shows that Cx47 level in treated knockout mice reached 38.73 ± 11.69% of the wild-type brain sample. KO = knockout.