Abstract
Standard immunogold-labeling methods in transmission electron microscopy (TEM) are unable to locate immunogold particles in the depth direction. This inability does not only concern bulky whole mounts, but also sections. A partial solution to the problem is stereo inspection. However, three-dimensional reconstruction of immunogold-labeled structures, that is, immuno-electron tomography (IET), is a correct solution for this inconsistency. Striking improvement in resolution is achieved: the 1.4-nm immunogold particles are shown in IET that are not detected in the original tilt series. IET is not restricted to laboratories with advanced medium- or high-voltage TEM and super-computing facilities; the methods we have developed for whole-mounted chromosomes and also for whole-mounted cytoskeleton of fibroblasts work remarkably well with ordinary 80-kV TEMs equipped with a goniometer to collect tilt series for IET on film. In addition, free programs are available to produce three-dimensional reconstructions even without high-performance computers. These improvements make it possible to many laboratories without modern facilities to perform IET reconstruction with standard TEM apparatus.
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References
Engelhardt, P. (2006) Three-dimensional reconstruction of chromosomes using electron tomography, in Electron Microscopy Methods and Protocols, 2nd ed (Kuo, J., ed.), Humana, Totowa, NJ, pp.365–385.
Engelhardt, P. (2000) Electron tomography of chromosome structure, in Encyclopedia of Analytical Chemistry (Meyers, R. A., ed.), vol. 6, John Wiley & Sons Ltd, Chichester, pp. 4948–4984. Available at: http://www.lce.hut.fi/~engelhar/. Accessed May 30, 2006.
Uchiyama, S., Kobayashi, S., Takata, H., et al. (2005) Proteome analysis of human metaphase chromosomes. J. Biol. Chem. 280, 16994–17004.
Stacey, G. (1998) Chick embryo fibroblast preparation, in Cell & Tissue Culture: Laboratory Procedures (Doyle, A., Griffiths, J. B., and Newell, D. G., eds.), John Wiley and Sons Ltd., England, pp. 3E:1.1–3E:1.3.
Meriläinen, J., Lehto, V. P., and Wasenius, V. M. (1997) FAP52, a novel, SH3 domain-containing focal adhesion protein. J. Biol. Chem. 272, 23278–23284.
Lehto, V. P. and Virtanen, I. (1985) Vinculin in cultured bovine lens-forming cells. Cell Differ. 16, 153–160.
Lewis, C. and Laemmli, U. (1982) Higher order metaphase chromosome structure: evidence for metalloprotein interactions. Cell 29, 171–181.
Spector, D., Goldman, R., and Leinwand, L. (1998) Chromosome isolation for biochemical and morphological analysis, in Cells: A Laboratory Manual (Janssen, K., ed.), vol. 1: Culture and Biochemical Analysis of Cells, Cold Springer Harbor Laboratory Press, New York, pp. 49.1–49.12.
Sone, T., Iwano, M., Kobayashi, S., et al. (2002) Changes in chromosomal surface structure by different isolation conditions. Arch. Histol. Cytol. 65, 445–455.
Uchiyama, S., Kobayashi, S., Takata, H., et al. (2004) Protein composition of human metaphase chromosomes analyzed by two-dimensional electrophoreses. Cytogenet. Genome Res. 107, 49–55.
Gasser, S. and Laemmli, U. (1987) Improved methods for the isolation of individual and clustered mitotic chromosomes. Exp. Cell Res. 173, 85–98.
Lucocq, J. (1992) Particulate markers for immunoelectron Microscopy, in Fine Structure Immunocytochemistry (Griffiths, G., ed.), Springer-Verlag, Berlin, pp. 279–305.
Slot, J. W. and Geuze, H. J. (1985) A new method for preparing gold probes for multiple-labelling cytochemistry. Eur. J. Cell Biol. 38, 87–93.
Gassmann, R., Henzing, A. J., and Earnshaw, W. C. (2004) Novel components of human mitotic chromosomes identified by proteomic analysis of the chromosome scaffold fraction. Chromosoma 113, 385–397.
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Engelhardt, P., Meriläinen, J., Zhao, F., Uchiyama, S., Fukui, K., Lehto, VP. (2007). Whole-Mount Immunoelectron Tomography of Chromosomes and Cells. In: Kuo, J. (eds) Electron Microscopy. Methods in Molecular Biology™, vol 369. Humana Press. https://doi.org/10.1007/978-1-59745-294-6_19
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DOI: https://doi.org/10.1007/978-1-59745-294-6_19
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