Influence of aldehyde fixation on the morphology of endosomes and lysosomes: quantitative analysis and electron tomography.

Détails

ID Serval
serval:BIB_F379CC0CC9BB
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Titre
Influence of aldehyde fixation on the morphology of endosomes and lysosomes: quantitative analysis and electron tomography.
Périodique
Journal of Microscopy
Auteur(s)
Murk J.L., Posthuma G., Koster A.J., Geuze H.J., Verkleij A.J., Kleijmeer M.J., Humbel B.M.
ISSN
0022-2720 (Print)
ISSN-L
0022-2720
Statut éditorial
Publié
Date de publication
2003
Volume
212
Numéro
Pt 1
Pages
81-90
Langue
anglais
Résumé
Cryoimmobilization is regarded as the most reliable method to preserve cellular ultrastructure for electron microscopic analysis, because it is both fast (milliseconds) and avoids the use of harmful chemicals on living cells. For immunolabelling studies samples have to be dehydrated by freeze-substitution and embedded in a resin. Strangely, although most of the lipids are maintained, intracellular membranes such as endoplasmic reticulum, Golgi and mitochondrial membranes are often poorly contrasted and hardly visible. By contrast, Tokuyasu cryosectioning, based on chemical fixation with aldehydes is the best established and generally most efficient method for localization of proteins by immunogold labelling. Despite the invasive character of the aldehyde fixation, the Tokuyasu method yields a reasonably good ultrastructural preservation in combination with excellent membrane contrast. In some cases, however, dramatic differences in cellular ultrastructure, especially of membranous structures, could be revealed by comparison of the chemical with the cryofixation method. To make use of the advantages of the two different approaches a more general and quantitative knowledge of the influence of aldehyde fixation on ultrastructure is needed. Therefore, we have measured the size and shape of endosomes and lysosomes in high-pressure frozen and aldehyde-fixed cells and found that aldehyde fixation causes a significant deformation and reduction of endosomal volume without affecting the membrane length. There was no considerable influence on the lysosomes. Ultrastructural changes caused by aldehyde fixation are most dramatic for endosomes with tubular extensions, as could be visualized with electron tomography. The implications for the interpretation of immunogold localization studies on chemically fixed cells are discussed.
Mots-clé
Aldehydes/chemistry, B-Lymphocytes/ultrastructure, Cell Line, Transformed, Cryopreservation/methods, Endosomes/ultrastructure, Freeze Substitution, Humans, Lysosomes/ultrastructure, Pressure, Tissue Fixation/methods, Tomography/methods, Tumor Cells, Cultured
Pubmed
Web of science
Création de la notice
18/10/2012 15:15
Dernière modification de la notice
03/03/2018 22:42
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