Analysis of acute brain slices by electron microscopy: A correlative light-electron microscopy workflow based on Tokuyasu cryo-sectioning.

Détails

Ressource 1Télécharger: BIB_7CA6BAFBE7CE.P001.pdf (3030.62 [Ko])
Etat: Public
Version: de l'auteur
Licence: Non spécifiée
ID Serval
serval:BIB_7CA6BAFBE7CE
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Institution
Titre
Analysis of acute brain slices by electron microscopy: A correlative light-electron microscopy workflow based on Tokuyasu cryo-sectioning.
Périodique
Journal of Structural Biology
Auteur(s)
Loussert Fonta C., Leis A., Mathisen C., Bouvier D.S., Blanchard W., Volterra A., Lich B., Humbel B.M.
ISSN
1095-8657 (Electronic)
ISSN-L
1047-8477
Statut éditorial
Publié
Date de publication
2015
Peer-reviewed
Oui
Volume
189
Numéro
1
Pages
53-61
Langue
anglais
Notes
Publication types: Journal Article Publication Status: ppublish
Résumé
Acute brain slices are slices of brain tissue that are kept vital in vitro for further recordings and analyses. This tool is of major importance in neurobiology and allows the study of brain cells such as microglia, astrocytes, neurons and their inter/intracellular communications via ion channels or transporters. In combination with light/fluorescence microscopies, acute brain slices enable the ex vivo analysis of specific cells or groups of cells inside the slice, e.g. astrocytes. To bridge ex vivo knowledge of a cell with its ultrastructure, we developed a correlative microscopy approach for acute brain slices. The workflow begins with sampling of the tissue and precise trimming of a region of interest, which contains GFP-tagged astrocytes that can be visualised by fluorescence microscopy of ultrathin sections. The astrocytes and their surroundings are then analysed by high resolution scanning transmission electron microscopy (STEM). An important aspect of this workflow is the modification of a commercial cryo-ultramicrotome to observe the fluorescent GFP signal during the trimming process. It ensured that sections contained at least one GFP astrocyte. After cryo-sectioning, a map of the GFP-expressing astrocytes is established and transferred to correlation software installed on a focused ion beam scanning electron microscope equipped with a STEM detector. Next, the areas displaying fluorescence are selected for high resolution STEM imaging. An overview area (e.g. a whole mesh of the grid) is imaged with an automated tiling and stitching process. In the final stitched image, the local organisation of the brain tissue can be surveyed or areas of interest can be magnified to observe fine details, e.g. vesicles or gold labels on specific proteins. The robustness of this workflow is contingent on the quality of sample preparation, based on Tokuyasu's protocol. This method results in a reasonable compromise between preservation of morphology and maintenance of antigenicity. Finally, an important feature of this approach is that the fluorescence of the GFP signal is preserved throughout the entire preparation process until the last step before electron microscopy.
Pubmed
Open Access
Oui
Création de la notice
14/01/2015 9:14
Dernière modification de la notice
15/07/2020 5:22
Données d'usage