Abstract
This chapter deals with tissue preparation for subsequent detection of molecules in biological samples using immunocytochemistry and transmission electron microscopy. The aim of these methods is to localize specific molecules at high resolution in order to identify their subcellular (or exact extracellular) localization. The methods are based on the use of antibodies or other affinity markers that bind specifically to a molecule of interest and a suitable detection system, e.g. a secondary antibody coupled to a gold particle of 5–15 nm size. Two different ways of sample preparation are described: (1) high-pressure freezing followed by freeze-substitution and immunogold labeling and (2) chemical fixation followed by freeze-substitution and immunogold labeling. Both methods have advantages and disadvantages that influence their utility in a given study design.
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References
Griffiths, G. (1993) Fine structure immunocytochemistry. Springer, Berlin, Germany.
Liou, W., Geuze, H.J., and Slot, J.W. (1996) Improving structural integrity of cryosections for immunogold labelling. Histochem. Cell Biol. 106, 41–58.
Skepper, J.N. (2000) Immunocytochemical strategies for electron microscopy: choice or compromise. J. Microsc. 199, 1–36.
Webster, P. and Webster, A. (2007) Cryosectioning fixed and cryoprotected biological material for immunocytochemistry. Methods Mol. Biol. 369, 257–289.
Robards, A.W. and Sleytr, U.B. (1985) In: Low temperature methods in biological electron microscopy. Elsevier, New York.
Moor, H. (1987) Theory and practice of high pressure freezing. In: Cryotechniques in biological electron microscopy. Edited by Steinbrecht R.A. and Zierold, K. Springer, Berlin, 175–191.
Shimoni, E. and Müller, M. (1998) On optimizing high-pressure freezing: from heat transfer theory to a new microbiopsy device. J. Microsc. 192, 236–247.
Studer, D., Graber, W., Al-Amoudi, A., and Eggli, P. (2001) A new approach for cryofixation by high-pressure freezing. J. Microsc. 203, 285–294.
Al-Amoudi, A., Norlen, L.P.O., and Dubochet, J. (2004) Cryo-electron microscopy of vitreous sections of native biological cells and tissues. J. Struct. Biol. 148, 131–135.
Mühlfeld, C., Rothen-Rutishauser, B., Vanhecke, D., Blank, F., Gehr, P., and Ochs, M. (2007) Visualization and quantitative analysis of nanoparticles in the respiratory tract by transmission electron microscopy. Part Fibre Toxicol. 4, 11.
McDonald, K.L., Morphew, M., Verkade, P., and Müller-Reichert, T. (2007) Recent advances in high-pressure freezing: equipment and specimen-loading methods. Methods Mol. Biol. 369, 143–173.
Tokuyasu, K.T. (1973) A technique for ultracryotomy of cell suspensions and tissues. J. Cell Biol. 57, 551–565.
Schwarz, H. and Humbel, B.M. (2007) Correlative light and electron microscopy using immunolabeled resin sections. Methods Mol. Biol. 369, 229–256.
Mühlfeld, C. and Richter, J. (2006) High-pressure freezing and freeze substitution of rat myocardium for immunogold labelling of connexin 43. Anat. Rec. A Discov. Mol. Cell. Evol. Biol. 288, 1059–1067.
Schmiedl, A., Ochs, M., Mühlfeld, C., Johnen, G., and Brasch, F. (2005) Distribution of surfactant proteins in type II pneumocytes of newborn, 14-day old, and adult rats: an immunoelectron microscopic and stereological study. Histochem. Cell Biol. 124, 465–476.
Brasch, F., Schimanski, S., Mühlfeld, C., Barlage, S., Langmann, T., Aslanidis, C., Boettcher, A., Dada, A., Schroten, H., Mildenberger, E., Prueter, E., Ballmann, M., Ochs, M., Johnen, G., Griese, M., and Schmitz, G. (2006) Alteration of the pulmonary surfactant system in full-term infants with hereditary ABCA3 deficiency. Am. J. Respir. Crit. Care Med. 174, 571–580.
Hagler, H.K. (2007) Ultramicrotomy for biological electron microscopy. Methods Mol. Biol. 369, 67–96.
Vanhecke, D., Graber, W., Herrmann, G., Al-Amoudi, A., Eggli, P., and Studer, D. (2003) A rapid microbiopsy system to improve the preservation of biological samples prior to high-pressure freezing. J. Microsc. 212, 3–12.
Hohenberg, H., Mannweiler, K., and Müller, M. (1994) High-pressure freezing of cell suspensions in cellulose capillary tubes. J. Microsc. 175, 34–43.
McDonald, K. (1999) High-pressure freezing for preservation of high resolution fine structure and antigenicity for immunolabeling. Methods Mol. Biol. 117, 77–97.
Monaghan, P., Perusinghe, N., and Müller, M. (1998) High-pressure freezing for immunocytochemistry. J. Microsc. 192, 248–258.
Buser, C. and Walther, P. (2008) Freeze-substitution: the addition of water to polar solvents enhances the retention of structures and acts at temperatures around –60°C. J. Microsc. 230, 268–277.
Hayat, M. (2000) Principles and techniques of electron microscopy. Cambridge University Press, Cambridge.
Mayhew, T.M. (2008) Taking tissue samples from the placenta: an illustration of principles and strategies. Placenta 29, 1–14.
Mühlfeld, C., Nyengaard, J.R., and Mayhew, T.M. A review of state-of-the-art stereology for better quantitative 3D morphology in cardiac research. Cardiovasc. Pathol. (epub ahead of print) doi: 10.1016/j.carpath. 2008.10.015)
Acknowledgements
The author wishes to thank Prof. Paul Webster, House Ear Institute, USA, for his very helpful suggestions to improve this manuscript and to Ms. Karola Michael for technical assistance with the preparation of the graphical work. The author is also thankful to the whole team of the EMBO Practical course “Electron microscopy and stereology in cell biology” held in Paris, September 2004, and the Faculty of Medicine, Georg-August-University Göttingen, whose research grant supported the study on high-pressure freezing of myocardium for immunolocalization of connexin 43.
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Mühlfeld, C. (2010). High-Pressure Freezing, Chemical Fixation and Freeze-Substitution for Immuno-electron Microscopy. In: Hewitson, T., Darby, I. (eds) Histology Protocols. Methods in Molecular Biology, vol 611. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-345-9_7
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DOI: https://doi.org/10.1007/978-1-60327-345-9_7
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