Eriochrome Cyanine R revisited: a standardized myelin stain protocol for thick sections of the central & peripheral...

Myelin enables rapid action potential conduction and axonal support, and its disruption underlies diverse neurological disorders. Its assessment is essential for studying development, plasticity, and repair of the nervous system (NS), and for diagnosing demyelinating diseases and evaluating remyelination therapies. Multiple histological methods detect myelin, each with trade-offs in sensitivity, cost, and reproducibility. Among them, Eriochrome Cyanine R (EC-R) is a simple, affordable myelin stain widely used in thin sections, but remains poorly standardized, and underexplored in thick vibratome sections. Here we describe and validate a simple, inexpensive, solvent-free EC-R protocol for central and peripheral nervous system tissue across seven vertebrate species and multiple demyelinating conditions. The method replaces subjective microscopic differentiation with fixed, time-controlled incubations scaled to section thickness, improving reproducibility. Using perfusion and immersion-fixed samples, we show that the protocol yields homogeneous myelin labeling with sharp white/gray matter contrast in whole brains, cortical slabs, spinal cord, and peripheral nerves. Thick sections stained with EC-R preserve 3-dimensional tissue architecture, resolve single myelinated axons and intracortical bands, and can be combined with Nissl-like counterstains and immunohistochemistry. Developmental series in neonatal rats reveal expected PNS-CNS myelination gradients, while experimental demyelination models and naturally occurring diseases (canine distemper and human multiple sclerosis) illustrate the method's ability to delineate lesion cores, perilesional gradients, and associated glial and immune activation. This standardized EC-R approach provides a robust and versatile tool for comparative neuroanatomy, experimental neuropathology, and translational studies of myelination, demyelination, and remyelination, as well as for the clinical diagnosis of myelin-related disorders.