Abstract
Detailed insight into the fine structure and 3D-architecture of the complex and dynamic compartments of the endocytic system is essential for a morpho-functional analysis of retrograde traffic from the cell surface to different intracellular destinations. Here, we describe a cytochemical approach for electron microscopic exploration of endocytic pathways with the use of wheat germ agglutinin (WGA) in combination with either conventional chemical fixation or ultrafast physical fixation of the cells by high pressure-freezing. Horseradish peroxidase-labeled WGA endocytozed by human hepatoma cells for various periods of time served as a marker. Its intracellular routes were visualized by means of diaminobenzidine oxidation either done conventionally after chemical fixation or in living cells prior to physical fixation. The latter protocol permits the combination of peroxidase-catalyzed cytochemistry with high pressure-freezing (HPF), which is state of the art for ultrastructural studies of complex and dynamic organelles at high spatial and temporal resolutions. The technique yields distinct cytochemical reactions and excellently preserved fine structures well qualified for detailed electron microscopic and 3D-studies of the complex endocytic architectures.
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Acknowledgements
The authors gratefully acknowledge the excellent technical assistance of Mag. Beatrix Mallinger, Mrs. Regina Wegscheider, Mr. Ulrich Kaindl, and Mr. Thomas Nardelli. Parts of this work were supported by the “Fonds zur Förderung der wissenschaftlichen Forschung” (FWF) grant P20116.
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Ranftler, C., Auinger, P., Meisslitzer-Ruppitsch, C., Ellinger, A., Neumüller, J., Pavelka, M. (2012). Electron Microscopy of Endocytic Pathways. In: Taatjes, D., Roth, J. (eds) Cell Imaging Techniques. Methods in Molecular Biology, vol 931. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-056-4_22
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DOI: https://doi.org/10.1007/978-1-62703-056-4_22
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