Tepon 812 epoxy resin

Mouse optic nerve samples were collected and 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, Hatfield, Pennsylvania) for a minimum of 48 hours. After fixation, samples were rinsed with 0.1M sodium cacodylate buffer, post-fixed with 2% osmium tetroxide in 0.1M sodium cacodylate buffer, then dehydrated with graded ethyl alcohol solutions, transitioned with propylene oxide and resin infiltrated in tEPON-812 epoxy resin (Tousimis, Rockville, Maryland) utilizing an automated EMS Lynx 2 EM tissue processor (Electron Microscopy Sciences, Hatfield, Pennsylvania). Processed tissues were oriented in tEPON-812 epoxy resin and polymerized in silicone molds using an oven set for 60°C for 48 hours. Semi-thin cross-sections were cut at 1-micron with a Histo diamond knife (Diatome, Hatfield, Pennsylvania) on a Leica UC-7 ultramicrotome (Leica Microsystems, Buffalo Grove, IL) and collected on slides then dried on a slide warmer. The slides were stained with 2% aqueous paraphenylenediamine (MP Biomedicals LLC, Solon, Ohio) solution for 45 minutes at room temperature, rinsed in tap and deionized water solutions, air-dried, then mounting medium and a glass coverslip was applied over the sections for light microscopic analysis of myelinated axon analysis.

Human specimens were fixed in 1/2 strength Karnovsky’s fixative and processed for TEM using standard procedures, as described previously^{70 (link), 71 (link)}. Briefly, specimens were rinsed in cacodylate buffer, post-fixed in 2% osmium tetroxide, en bloc stained with 2% uranyl acetate, dehydrated with alcohol to propelene oxide, and infiltrated with tEPON-812 epoxy resin (Tousimis, Rockville, MD) utilizing an automated EMS Lynx 2 EM tissue processor (Electron Microscopy Sciences, Hatfield, PA). Ultrathin sections (80 nm) were cut from the epoxy block using a Leica EM UC7 ultramicrotome (Leica Microsystems, Buffalo Grove, IL) and a diamond knife. Sections were imaged using an FEI Tecnai G2 Spirit transmission electron microscope (FEI, Hillsboro, Oregon) at 80 kV interfaced with an AMT XR41 digital CCD camera (Advanced Microscopy Techniques, Woburn, MA).