Myelin Histology

Surgical and histology procedures were in accordance with European requirements (86/609/EEC) and approved by the appropriate veterinary and ethical services. The experiment was conducted on a single Cynomolgus macaque (Macaca fascicularis, 15 year-old female, 5.2 kg). The detailed protocol for surgery, euthanasia and perfusion can be found elsewhere (Markov et al., 2014). The myelin stain was performed on mounted sections by adjusting the modified Gallyas silver stain method of which the details can be found in Pistorio et al, 2006. Briefly, after perfusion and extraction, the brain was cut in 40 μm-thick sections on a freezing microtome. 1 out of 24 sections (960 μm interval) were mounted on 3% gelatin-coated slices for myelin stain. After drying overnight, myelin silver staining begins with a fixation in formalin, followed by acetylation in 2:1 pyridine and acetic anhydride mixture and successive baths of decreasing concentration of pyridine. Those steps efficiently reduce the ability of the other tissue elements to retain staining, mainly cell nuclei, and ensure that myelin is the only compound that can effectively bind with silver particles. After rinsing with water, sections were bathed in silver nitrate solution for impregnation, the surplus of silver is then washed away by 0.5% acetic acid, followed by re-fixation in formalin. After the re-fixation, the sections are immersed in developer solution, thus revealing myelin fibres. This step is critical and must be visually monitored by experimentalists under a dissecting microscope. When the appropriate contrast is reached, the revelation reaction is stopped with 1% acetic acid, and histological contrast is adjusted by differentiation (i.e. destaining) of overdeveloped sections with 0.1% potassium ferricyanide solution. Sections are then washed by 0.5% sodium thiosulfate and water, dehydrated and coverslipped.