Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
2178678 | European Journal of Cell Biology | 2010 | 13 Pages |
Abstract
Immunolabelling of (ultra)thin thawed cryosections according to Tokuyasu is one of the most reliable and efficient immunolocalisation techniques for cells and tissues. However, chemical fixation at ambient temperature, a prerequisite of this technique, can cause problems for samples, like plant tissue, because cell walls, hydrophobic surfaces and intercellular air slow down diffusion of fixative molecules into the sample. We show that a hybrid technique, based on a combination of cryofixation/freeze-substitution and Tokuyasu cryosection immunolabelling, circumvents the disadvantages associated with chemical fixation and results in an improved ultrastructure and antigenicity preservation of Tokuyasu cryosections used for light and electron microscopic immunolabelling (as shown for Myc- or mRFP-tagged proteins, KNOLLE and carbohydrate epitopes). In combination with the most sensitive particulate marker systems, like 1-nm gold or quantum dot markers, we were able to obtain a differentiated labelling pattern which allows a more detailed evaluation of plant Golgi, trans-Golgi network and multivesicular body/prevacuolar compartment markers (COPI-specific γCOP, the ADP-ribosylation factor GTPase ARF1, ARA7/RabF2b and the vacuolar sorting receptor VSR). We also discuss possibilities to improve membrane contrast, e.g., of transport vesicles like COPI, COPII and clathrin-coated vesicles, and of compartments of endosomal trafficking like the trans-Golgi network.
Keywords
Related Topics
Life Sciences
Agricultural and Biological Sciences
Plant Science
Authors
York-Dieter Stierhof, Farid El Kasmi,