A method to prepare large resin sections for counting myelinated axons in rodent CNS and PNS structures

•1−3 μm thick transversal sections can be prepared from the resin-embedded specimens as large as rat brain hemisphere.•Stereological methods, identification and counting of individual myelinated axons can be applied using this protocol.•Neurocounter – free custom-made software is an optimal tool for semi-automated quantification of axons. We present a method that allows preparing histological sections from large blocks of nervous tissue embedded in epoxy resin. Resin-embedding provides excellent resolution especially for the myelin-rich white matter and is often being used for visualizing the myelinated axons in peripheral nerves. However, because of the limited penetration of the reagents, only very small tissue specimens can be processed in this way. Here, we describe a method that enables to embed large specimens and their sectioning on a standard sliding microtome. To process the large specimens, modifications in several steps of the processing technique had to be made. In this paper we demonstrate, that with this technique 1−3 μm thick transversal sections can be prepared from the resin-embedded specimens as large as rat brain hemisphere. Such a large section allows simultaneously: 1.) overviewing and delineating the gross anatomical structures, and 2.) observing the subcellular details at the highest possible optical magnifications. Such a large section with excellent resolution allows application of unbiased stereological methods and reliable quantification of very small objects within the area of interest.