Item v 5

The optic nerves of transcardially perfused mice were postfixed overnight in 4% PFA and 2% glutaraldehyde in cacodylate buffer. Nerves were osmicated and processed for light and electron microscopy; morphometric quantification of neuropathological alterations was performed as published previously^{29 (link)} using a LEO906 E electron microscope (ZEISS) and corresponding software iTEM v.5.1 (Soft Imaging System). At least 10 regions of interest (corresponding to an area of around 5% and up to 3000 axons per individual optic nerve) were analyzed per optic nerve per mouse. The percentages of axonal profiles showing spheroid formation or undergoing degeneration were identified individually by their characteristic morphological features in electron micrographs and related to the number of all investigated axons per optic nerve per mouse. Images were processed (rotation, cropping, addition of symbols and pseudocolor) to generate figures using Photoshop CS6 and Illustrator CS6 (Adobe).

The optic nerves of transcardially perfused mice were postfixed overnight in 4% PFA and 2% glutaraldehyde in cacodylate buffer. Nerves were osmicated and processed for light and electron microscopy; morphometric quantification of neuropathological alterations was performed as published previously¹⁸ (link) using a LEO906 E electron microscope (ZEISS) and corresponding software iTEM v.5.1 (Soft Imaging System). At least 10 regions of interest (corresponding to an area of around 5% and up to 3,000 axons per individual optic nerve) were analyzed per optic nerve per mouse. The percentages of axonal profiles showing spheroid formation or undergoing degeneration were identified individually by their characteristic morphological features in electron micrographs and related to the number of all investigated axons per optic nerve per mouse. Total axon counts were estimated by relating the total axonal densityto the cross-sectional area of the respective optic nerve section. Genetically perturbed myelin in heterozygous females was identified by myelin compaction defects at high resolution. Images were processed (rotation, cropping, addition of symbols and pseudocolor) to generate figures using Photoshop CS6.