High efficiency labeling of nerve fibers in cleared tissue for light-sheet microscopy
Year: 2025
Authors: Rojas-Rodriguez M., Imbimbo E., Capitini C., Costantini I., Mazzamuto G., Pavone F.S., Silvestri L., Calamai M.
Autors Affiliation: European Lab Nonlinear Spect, Via Nello Carrara 1, I-50019 Sesto Fiorentino, Italy; Univ Florence, Dept Phys & Astron, Via G Sansone 1, I-50019 Sesto Fiorentino, Italy; Natl Res Council CNR INO, Natl Inst Opt, I-50125 Sesto Fiorentino, Italy; Univ Florence, Dept Biol, I-50019 Sesto Fiorentino, Italy.
Abstract: Background: Tissue clearing techniques combined with light-sheet fluorescence microscopy (LSFM) enable highresolution 3D imaging of biological structures without physical sectioning. While widely used in neuroscience to determine brain architecture and connectomics, their application for spinal cord mapping remains more limited, posing challenges for studying demyelinating diseases like multiple sclerosis. Myelin visualization in cleared tissues is particularly difficult due to the lipid-removal nature of most clearing protocols, and alternative immunolabeling approaches failed to reach satisfying results. New method: To overcome these limitations, we developed a novel protocol named HELF -High Efficiency Labeling of Fibers- which takes advantage of a fluorescently labeled aminosterol, trodusquemine, which displays a strong affinity for cholesterol-rich membranes, and a supplementary round of fixation with glutaraldehyde. Results and comparison with existing methods: The labeling with trodusquemine was tested in combination with various established tissue clearing techniques and compared with HELF, which resulted to be the best approach for providing high-brightness myelin staining in mouse spinal cord and brain, and in human brain samples. Finally, we demonstrated that HELF can be used to stain and image with LSFM a whole cleared mouse spinal cord. Conclusions: Our data support the potential use of HELF coupled to LSFM as a practical tool for the evaluation of novel therapeutics for remyelination in preclinical models of CNS diseases.
Journal/Review: JOURNAL OF NEUROSCIENCE METHODS
Volume: 424 Pages from: 110567-1 to: 110567-10
More Information: LS was supported by #NEXTGENERATIONEU (NGEU) and funded by Italian Ministry of University and Research (MUR) under the PRIN 2022 program (2022HY8KXW-Deciphering the whole-brain circuit mediating traumatic memory attenuation) and under the National Recovery and Resilience Plan (NRRP), project MNESYS (PE0000006)-A multi-scale integrated approach to the study of the nervous system in health and disease (DN. 15 53 11.10.2022); he was also funded by the Italian Ministry of Foreign Affairs and International Cooperation, grant n.PGR02066 (Strategic Italy-US projects).KeyWords: Spinal cord; LSFM; Nerve fibers; Trodusquemine; Myelin; Tissue clearing. (Min.1-Max. 7)DOI: 10.1016/j.jneumeth.2025.110567
