Handling Difficult Cryo-ET Samples: A Study with Primary Neurons from Drosophila melanogaster.

Autor:	Kim JY; Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.; Department of Chemistry, University of Wisconsin-Madison, Madison, WI 53706, USA., Yang JE; Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.; Cryo-Electron Microscopy Research Center, University of Wisconsin-Madison, Madison, WI 53706, USA.; Midwest Center for Cryo-Electron Tomography, Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA., Mitchell JW; Department of Chemistry and Biochemistry, Kalamazoo College, Kalamazoo, MI 49006, USA., English LA; Neuroscience Training Program, University of Wisconsin-Madison, Madison, WI 53705, USA., Yang SZ; Department of Biology, Stanford University, Stanford, CA 94305, USA., Tenpas T; Department of Neuroscience, University of Wisconsin-Madison, Madison, WI 53705, USA., Dent EW; Department of Neuroscience, University of Wisconsin-Madison, Madison, WI 53705, USA., Wildonger J; Departments of Pediatrics and Biological Sciences, University of California, San Diego, La Jolla, CA 92093, USA., Wright ER; Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.; Cryo-Electron Microscopy Research Center, University of Wisconsin-Madison, Madison, WI 53706, USA.; Midwest Center for Cryo-Electron Tomography, Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.; Morgridge Institute for Research, University of Wisconsin-Madison, Madison, WI 53715, USA.
Jazyk:	angličtina
Zdroj:	Microscopy and microanalysis : the official journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada [Microsc Microanal] 2023 Dec 21; Vol. 29 (6), pp. 2127-2148.
DOI:	10.1093/micmic/ozad125
Abstrakt:	Cellular neurobiology has benefited from recent advances in the field of cryo-electron tomography (cryo-ET). Numerous structural and ultrastructural insights have been obtained from plunge-frozen primary neurons cultured on electron microscopy grids. With most primary neurons having been derived from rodent sources, we sought to expand the breadth of sample availability by using primary neurons derived from 3rd instar Drosophila melanogaster larval brains. Ultrastructural abnormalities were encountered while establishing this model system for cryo-ET, which were exemplified by excessive membrane blebbing and cellular fragmentation. To optimize neuronal samples, we integrated substrate selection, micropatterning, montage data collection, and chemical fixation. Efforts to address difficulties in establishing Drosophila neurons for future cryo-ET studies in cellular neurobiology also provided insights that future practitioners can use when attempting to establish other cell-based model systems. Competing Interests: Conflict of Interest The authors declare that they have no competing interest. (© The Author(s) 2023. Published by Oxford University Press on behalf of the Microscopy Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)
Databáze:	MEDLINE
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