Visualizing the synaptic and cellular ultrastructure in neurons differentiated from human induced neural stem cells - an optimized protocol

Please always quote using this URN: urn:nbn:de:bvb:20-opus-236053
  • The size of the synaptic subcomponents falls below the limits of visible light microscopy. Despite new developments in advanced microscopy techniques, the resolution of transmission electron microscopy (TEM) remains unsurpassed. The requirements of tissue preservation are very high, and human post mortem material often does not offer adequate quality. However, new reprogramming techniques that generate human neurons in vitro provide samples that can easily fulfill these requirements. The objective of this study was to identify the cultureThe size of the synaptic subcomponents falls below the limits of visible light microscopy. Despite new developments in advanced microscopy techniques, the resolution of transmission electron microscopy (TEM) remains unsurpassed. The requirements of tissue preservation are very high, and human post mortem material often does not offer adequate quality. However, new reprogramming techniques that generate human neurons in vitro provide samples that can easily fulfill these requirements. The objective of this study was to identify the culture technique with the best ultrastructural preservation in combination with the best embedding and contrasting technique for visualizing neuronal elements. Two induced neural stem cell lines derived from healthy control subjects underwent differentiation either adherent on glass coverslips, embedded in a droplet of highly concentrated Matrigel, or as a compact neurosphere. Afterward, they were fixed using a combination of glutaraldehyde (GA) and paraformaldehyde (PFA) followed by three approaches (standard stain, Ruthenium red stain, high contrast en-bloc stain) using different combinations of membrane enhancing and contrasting steps before ultrathin sectioning and imaging by TEM. The compact free-floating neurospheres exhibited the best ultrastructural preservation. High-contrast en-bloc stain offered particularly sharp staining of membrane structures and the highest quality visualization of neuronal structures. In conclusion, compact neurospheres growing under free-floating conditions in combination with a high contrast en-bloc staining protocol, offer the optimal preservation and contrast with a particular focus on visualizing membrane structures as required for analyzing synaptic structures.show moreshow less

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Metadaten
Author: Philipp CapetianORCiD, Lorenz Müller, Jens Volkmann, Manfred Heckmann, Süleyman Ergün, Nicole Wagner
URN:urn:nbn:de:bvb:20-opus-236053
Document Type:Journal article
Faculties:Medizinische Fakultät / Institut für Anatomie und Zellbiologie
Medizinische Fakultät / Physiologisches Institut
Medizinische Fakultät / Neurologische Klinik und Poliklinik
Language:English
Parent Title (English):International Journal of Molecular Sciences
ISSN:1422-0067
Year of Completion:2020
Volume:21
Issue:5
Article Number:1708
Source:International Journal of Molecular Sciences (2020) 21:5, 1708. https://doi.org/10.3390/ijms21051708
DOI:https://doi.org/10.3390/ijms21051708
Dewey Decimal Classification:6 Technik, Medizin, angewandte Wissenschaften / 61 Medizin und Gesundheit / 610 Medizin und Gesundheit
Tag:high contrast; human neurons; induced neural stem cells; synapse; synaptic vesicles; transmission electron microscopy
Release Date:2021/05/10
Open-Access-Publikationsfonds / Förderzeitraum 2020
Licence (German):License LogoCC BY: Creative-Commons-Lizenz: Namensnennung 4.0 International