Background

Myelin is the multilayered, molecularly specialized plasma membrane of oligodendrocytes in the CNS or Schwann cells in the peripheral nervous system that accelerates nerve conduction by ensheathing axons (for comprehensive reviews: Hildebrand et al., 1993; Kidd et al., 2013; Nave and Werner, 2014; Monk et al., 2015; Simons and Nave, 2015). The myelination of axons provides electrical insulation and thus facilitates saltatory impulse propagation in a highly efficient manner (Tasaki, 1939; Hartline and Colman, 2007). In addition to insulation, myelinating cells may provide trophic support for myelinated axons (Lappe-Siefke et al., 2003; Nave, 2010; Funfschilling et al., 2012; Joseph et al., 2019).

Electron microscopy reveals morphologically distinguishable subcompartments of myelin. The compacted myelin comprises alternating electron-dense and electron-lucent layers while the adaxonal myelin layer and paranodal myelin loops remain non-compacted. At the light microscopic level, large myelinated tracts (i.e., the white matter including the corpus callosum) are clearly distinguishable from the grey matter, in which the majority of axons is non-myelinated. However, even largely unmyelinated CNS regions including the cortex comprise individual myelinated axons that appear as fine fibers if visualized by specific staining.

Ferenc Gallyas described in 1979 a method to specifically visualize myelin in the brain (Gallyas, 1979). A fundamental step is the treatment of the CNS tissue with pyridine and acetic anhydride, which during the subsequent silver impregnation prevents the absorption of silver ions by tissues other than myelin. Another critical step is the physical developer, which contains silver nitrate and tungstosilicic acid, which, once added, reduces silver ions to elemental silver. In 2005, Pistorio and colleagues described a modified version of this method by Gallyas (Pistorio et al., 2006). Here, we provide our lab protocol based on these previous versions. The major difference is the use of a microwave to decrease the staining time as heat causes the molecules to diffuse rapidly across tissues. Other differences include a slight modification in the recipe of the physical developer and the absence of a de-staining step involving potassium ferricyanide. In recent years, silver impregnation of myelin has been mainly used to assess myelinated tracts in mouse mutants (de Monasterio-Schrader et al., 2012; Werner et al., 2013; Patzig et al., 2016; Erwig et al., 2019; Joseph et al., 2019), yet the method can be applied to various species and scientific questions. As we describe the silver impregnation of paraffinated brain sections, we included the paraffin embedding and the de-paraffinization procedure.