Figure 10. Impaired trafficking of MAG in Pikfyveflox/flox,Olig2Cre brain slices.
Confocal images of acute brain slices in oxygenated ACSF treated with bath-applied anti-MAG-Alexa555 antibody, fixed and stained with anti-mouse-Alexa488 secondary antibody to distinguish between endocytosed MAG (red) and PM localized MAG (green). (A) OLs in the striatum of Pikfyve control mice (P13) show punctate MAG labeling in the cell soma (arrows) and along processes that form internodes. Only few MAG+ structures are also stained with anti-mouse-Alexa488, and thus, localized on the PM. (B-B”) Limited perinuclear MAG labeling is observed in the Pikfyveflox/flox,Olig2Cre striatum. Many MAG+ structures are labeled red and green, and thus localized to the PM, however intracellular MAG is observed in some cells. Scale bar = 20 µm. Small inset shows a 3D view of the two cells labeled with arrows (B-B”). MAG+ vesicles (red) only partially overlap with PM localized MAG (green). Alexa488+ isosurface transparency is adjusted to 50% to demonstrate intracellular Alexa555+ (red) and LiveNuc 647+ (blue) structures. Scale bar = 10 µm. To directly demonstrate that anti-MAG antibody labeled cells are OLs, parallel experiments were carried out with brain slices of LacZ/EGFP,Olig2Cre reporter mice. Anti-MAG antibody specificity is demonstrated with Mag-/-slices in Figure 10—figure supplement 1.
Figure 10—figure supplement 1. Anti-MAG labeling of EGFP+ OLs and specificity control for anti-MAG antibody in acute brain slices.
