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Updated: Sep 19, 2025
En face Cryosectioning of Mouse Retina for High-dimensional Spatial Molecular Analysis
Published on: July 8, 2025
Protocol for assessing regional pathology in the rodent optic nerve using longitudinal cryosections and
Gabrielle M Mey1, John Shelestak2, Muhammad Irfan2
1Department of Ophthalmology and Visual Sciences, University of Michigan, Michigan Medicine, Ann Arbor, MI 48105, USA; Michigan Neuroscience Institute, University of Michigan, Michigan Medicine, Ann Arbor, MI 48105, USA.
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Visual system pathology has been established as a prominent feature in demyelinating diseases such as multiple sclerosis (MS). Here, we present a protocol for assessing regional pathology in the rodent optic nerve using longitudinal cryosections and cross-sectional electron microscopy. We describe steps for optic nerve dissection, post-fixation, myelin detection, slide scanning of myelin basic protein, and the analysis of demyelination. We then detail procedures for fluorescent antibody staining, analysis for transmission electron microscopy (TEM), and the analysis of myelinated and unmyelinated axons. For complete details on the use and execution of this protocol, please refer to Mey et al.1,2.
