Abstract
Multiple label immunoelectron microscopy localizes and detects multiple antigens in cells and tissues. In double labeling, two kinds of primary antibodies from different animal species are used after being mixed in a single solution. To distinguish the different antigens, secondary antibodies should be labeled with colloidal gold particles of different diameter. Generally, the secondary antibody that is used for detecting the antigen with lower distribution density is labeled with smaller-sized gold particles. In this chapter, double-label immunoelectron microscopy of gelatin-embedded cultured cells using the cryosectioning technique is described.
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References
Tokuyasu, K. T. (1978) A study of positive staining of ultrathin frozen sections. J. Ultrastruct. Res. 63, 287–307.
Tokuyasu, K. T. (1986) Application of cryoultramicrotomy to immunocytochemistry. J. Microsc. 143, 139–149.
Tokuyasu, K. T. (1989) Use of poly(vinylpyrrolidone) and poly(vinyl alcohol) for cryoultramicrotomy. Histochem. J. 21, 163–171.
Liou, W., Geuze, H. J., and Slot, J. W. (1996) Improving structural integrity of cryosections for immunogold labeling. Histochem. Cell Biol. 106, 41–58.
Webster, P. and Webster, A. (2007) Cryosectioning fixed and cryoprotected biological material for immunocytochemistry. Methods Mol. Biol. 369, 257–289.
Fujimoto, T., Tokuyasu, K. T., and Singer, S. J. (1987) Direct morphological demonstration of the coexistence of vimentin and desmin in the same intermediate filaments of vascular smooth muscle cells. J. Submicrosc. Cytol. 19, 1–9.
Aoki, T., Hagiwara, H., Matsuzaki, T., Suzuki, T., and Takata, K. (2007) Internalization of caveolae and their relationship with endosomes in cultured human and mouse endothelial cells. Anat. Sci. Int. 82, 82–97.
Acknowledgments
This work was supported, in part, by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, & Technology of Japan.
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Hagiwara, H., Aoki, T., Suzuki, T., Takata, K. (2010). Double-Label Immunoelectron Microscopy for Studying the Colocalization of Proteins in Cultured Cells. In: Schwartzbach, S., Osafune, T. (eds) Immunoelectron Microscopy. Methods in Molecular Biology, vol 657. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-783-9_20
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DOI: https://doi.org/10.1007/978-1-60761-783-9_20
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Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60761-782-2
Online ISBN: 978-1-60761-783-9
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