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          Institute: MPI für Entwicklungsbiologie     Collection: Abteilungsunabhängige Arbeitsgruppen     Display Documents



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ID: 332609.0, MPI für Entwicklungsbiologie / Abteilungsunabhängige Arbeitsgruppen
Cryo-section immunolabelling of difficult to preserve specimens: advantages of cryofixation, freeze-substitution and rehydration.
Authors:Ripper, D.; Schwarz, Heinz; Stierhof, York-Dieter
Language:English
Date of Publication (YYYY-MM-DD):2008-02
Title of Journal:Biol Cell. 2008 Feb;100(2):109-23.
Volume:100
Issue / Number:2
Start Page:109
End Page:23
Sequence Number of Article:17903123
Review Status:not specified
Audience:Not Specified
Abstract / Description:BACKGROUND INFORMATION: Electron microscopic immunolabelling of ultrathin thawed cryo-sections, according to the method of Tokuyasu, is widely used as a very sensitive high-resolution localization technique. Its main advantages are that antigens remain in a hydrated environment prior to immunolabelling, and that antigen accessibility is improved compared with resin section labelling. However, the quality of structural appearance and antigenicity depends highly on the limitations of the initial conventional chemical fixation step, such as slow diffusion and selective reaction/cross-linking of fixative molecules. RESULTS AND CONCLUSIONS: Cryofixation, instead of conventional chemical fixation, followed by freeze-substitution/chemical fixation, rehydration and further processing for Tokuyasu cryo-sectioning leads to an improved preservation of both ultrastructure and antigenicity. This is especially true for tissues which are difficult to preserve by conventional chemical fixation at ambient temperatures, such as plant material, Drosophila embryos or nematode tissue. In particular labile and highly dynamic structures (for example, microtubules and Golgi apparatus) are remarkably better preserved. These improvements are also valid for light microscopic applications.
External Publication Status:published
Document Type:Article
Affiliations:MPI für Entwicklungsbiologie/Abteilungsunabhängige Arbeitsgruppen
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