Optic nerve cross sections were processed using a modified paraphenylenediamine (PPD) staining protocol to stain the myelin sheath of all axons and the axoplasma of damaged axons, as previously described (66 (link)). Optic nerves were fixed with half-strength Karnovsky’s fixative [2% formaldehyde + 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4 (Electron Microscopy Sciences)] for a minimum of 24 h at 40 °C. After fixation, samples were rinsed with 0.1 M sodium cacodylate buffer, postfixed with 2% osmium tetroxide in 0.1 M sodium cacodylate buffer, dehydrated with graded ethyl alcohol solutions, and transitioned with propylene oxide and resin infiltrated in tEPON-812 epoxy resin (Tousimis). Optic nerve cross-sections (1 μm) were stained with filtered 2% aqueous PPD (MP Biomedicals, LLC) solution for 1 h at room temperature, rinsed in tap and deionized water solutions, and air-dried. Mounting medium and a glass coverslip were applied over the sections for light microscopic analysis.