Abstract
The immunolocalization of proteins on sections of biological materials that have been prepared for electron microscopy, requires the preservation of both antigenic and morphological structures. In this chapter, two techniques that fulfill these requirements, often difficult to reconcile, but that have been used extensively for mammalian cells, will be described in their adaptation for yeast cells: the progressive low temperature embedding technique in Lowicryl introduced by Carlemalm et al. (1) and cryo-ultramicrotomy as described by Tokuyasu (2). The immunolocalization technique used in this chapter is based on the protein-A gold technique described by Roth et al. (3) and Geuze et al. (23).
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Carlemalm, E., Garavito, M., and Villiger, W. (1982) Resin development for electron microscopy and an analysis of embedding at low temperature. J. Microsc. 126, 123–143.
Tokuyasu, K. T. (1973) A technique for ultracryotomy of cell suspensions and tissues. J. Cell Biol. 57, 551–565.
Roth, J., Bendayan, M., Carlemalm, E., Villiger, E., and Garavito, M. (1981) Enhancement of structural presentation and immununocytochemical staining in low temperature embedded pancreatic tissue. J. Histochem. Cytochem. 29, 663–671.
Geuze, H. J., Slot, J. W. van der Ley, P. A., and Scheffer, R. C. T. (1981) Use of colloidal gold particles in double-labelling immununoelectron microscopy of frozen tissue sections. J. Cell Biol. 89, 653–665.
van Tuinen, E. and Riezman, H. (1987) Immunolocalization of glyceraldehyde-3-phosphate dehydrogenase, hexokinase and carboxypeptidase Y in yeast cells at the ultrastructural level. J. Histochem. Cytochem. 35, 327–333.
Tokuyasu, K. T. and Singer, S. J. (1976) Improved procedures for immunoferritin labelling of ultrathin frozen sections. J. Cell Biol. 71, 894–906.
Griffith, G., Brands, R., Burke, B., Louvard, D., and Warren, G. (1982) Viral membrane proteins acquire galactose in trans golgi cisternae during intracellular transport. J. Cell Biol. 95, 781–792.
Griffiths, G., McDowall, A., Back, R., and Dulbochet, J. (1984) On the preparation of cryosections of immunocytochemistry. J. Ultrastruct. Res. 89, 65–78.
Roth, J. (1989) Postembedding labelling on Lowicryl K4M tissue sections, detection and modification of cellular components, in Meth. Cell Biol. 31, 513–551.
Griffith, G., Simons, K., Warren, G., and Tokuyasu, K. T. (1983) Immunoelectron microscopy using thin, frozen sections: application to studies of the intracellular transport of semliki forest virus spike glyroproteins. Methods Enzymol. 96, 466–483.
Slot, J. W. and Geuze, H. J. (1985) A new method of preparing gold probes for multiple-labelling cytochemistry. Eur. J. Cell Biol. 38, 87–93.
Slot, J. W., Geuze, H. J., and Weerkamp, A. H. (1988) Localization of macromolecular components by application of the immunogold technique on cryosectioned bacteria. Methods Microbiol. 20, 211–236.
Millonig, G. (1961) A modified procedure for lead staining of thin sections. J. Biophys. Biochem. 11, 736–739.
Stevens, B. J. (1977) Variation in number and volume of the mitochondria in yeast according to growth conditions. Biol. Cellulair 28, 37–56.
Stevens, B. (1981) The molecular biology of the yeast Saccharomyces: life cycle and inheritance. (Strathern, J. N., Jones, E. W., and Broach, J. R., eds.), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 471–504.
Byers, B. and Goetsch, L. (1975) Behaviour of spindles and spindle plaques in the cell cycle and conjugation of Saccharomyces cerevisiae. J. Bacteriol. 24, 511–523.
Byers, B. (1982) Cytology of the yeast life cycle, in The Molecular Biology of the Yeast Saccharomyces. Life Cycle and Inheritance (Strathern, J. N., Jones, E. W., and Broach, J. R., eds.), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 59–96.
Wright, R. and Rine, J. (1989) Transmission Electron Microscopy and immunocytochemical Studies of Yeast, in Methods in Cell Biology, Academic, San Diego, CA, vol. 31, pp. 473–512.
Glauert, M. (1974) Practical Methods in Electron Microscopy, vol. 3, North Holland, Amsterdam, pp. 1–110.
Griffith, G. and Hoppeler, H. (1986) Quantitation in immunocyto-chemistry: correlation of immunogold labelling to absolute number of membrane antigens. J. Histochem. Cytochem. 34, 1389–1398.
Kellenberger, E., Durrenberger, M., Villiger, W., Carlemalm, E., and Wurtz, M. (1987) The efficiency of immunolabel on Lowicryl sections compared to theoretical predictions. J. Histochem Cytochem. 35, 959–969.
Stevens, T. H., Rotheman, J. H., Payne, G. S., and Schekman, R. (1986) Gene dosage-dependent secretion of yeast vacuolar carboxypeptidase Y. J. Cell Biol. 102, 1551–1557.
Slot, J. W. and Geuze, H. (1984) Gold markers of single and double immunolabelling of ultrathin cryosections, in, Immunolabeling for electron microscopy (Polak, J. M. and Varndell, I. M., eds.), Elsevier, Amsterdam, pp. 129–142.
Webster, D. L. and Watson, K. (1993) Ultrastructural changes in yeast following heat shock and recovery. Yeast 9, 1165–1175.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1996 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
van Tuinen, EJ. (1996). Immunoelectron Microscopy. In: Evans, I.H. (eds) Yeast Protocols. Methods in Molecular Biology™, vol 53. Humana Press. https://doi.org/10.1385/0-89603-319-8:407
Download citation
DOI: https://doi.org/10.1385/0-89603-319-8:407
Publisher Name: Humana Press
Print ISBN: 978-0-89603-319-1
Online ISBN: 978-1-59259-540-2
eBook Packages: Springer Protocols