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Electron Microscopy pp 273–313Cite as

Cryosectioning Fixed and Cryoprotected Biological Material for Immunocytochemistry

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1117))

Abstract

Immunocytochemistry for electron microscopy provides important information on the location and relative abundance of proteins inside cells. Gaining access to this information without extracting or disrupting the location of target proteins requires specialized preparation methods. Sectioning frozen blocks of chemically fixed and cryoprotected biological material is one method for obtaining immunocytochemical data. Once the cells or tissues are cut, the cryosections are thawed, mounted onto coated grids, and labeled with specific antibodies and colloidal gold probes. They are then embedded in a thin film of plastic containing a contrasting agent. Subcellular morphology can then be correlated with specific affinity labeling by examination in the transmission electron microscope (TEM). The major advantage of using thawed cryosections for immunolabeling is that the sections remain fully hydrated through the immunolabeling steps, reducing the possibility of dehydration-induced antigen modification. Modern technical advancements both in preparation protocols and equipment design make cryosectioning a routine and rapid approach for immunocytochemistry that may provide increased sensitivity for some antibodies.

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Acknowledgements

This simple set of instructions for preparing cryosections represents the combined efforts of many people. Certainly, the innovative work of Professor K. Tokyuasu made it possible to section frozen biological material while retaining ultrastructure and antigenicity. However, this innovation would not have been possible without the apparatus built by A. Kent Christensen. He built the first cryosectioning box able to fit on an ultramicrotome in the early 1970s. However, cryosectioning was first attempted by Fernandez-Moran in the 1950s [14], who was also the first to introduce the diamond knife for ultrathin sectioning [8–10].

Recognizing the power of cryosectioning and immunocytochemistry for cellular biology, Jan W. Slot and Hans J. Geuze (Utrecht, the Netherlands) and Gareth Griffiths (EMBL in Heidelberg, Germany) with Professor K. T. Tokuyasu in San Diego incorporated the method into their work. These pioneers worked closely to understand the cryosectioning method and introduce technical improvements. Most importantly they made the method available to the scientific community by teaching it on courses organized in their own laboratories and on courses sponsored by the European Molecular Biology Organization (EMBO), and on courses throughout the world.

EMBO has been generous in funding training courses that have introduced young scientists to immunocytochemical methods using cryosections. One important and unplanned result of the EMBO courses has been their effect on technique development. Bringing together a group of scientists together for 10 days each year has resulted in innovative developments and rapid spread of these innovations. This chapter was originally developed as a teaching aid for EMBO practical courses and is still used on these courses.

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Authors and Affiliations

  1. Center for Electron Microscopy and Microanalysis (CEMMA), University of Souther California, Los Angeles, CA, USA

    Paul Webster

  2. Division of Biology, California Institute of Technology, Pasadena, CA, USA

    Alexandre Webster

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  1. Paul Webster
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  1. University of Western Australia, Crawley, Australia

    John Kuo

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Webster, P., Webster, A. (2014). Cryosectioning Fixed and Cryoprotected Biological Material for Immunocytochemistry. In: Kuo, J. (eds) Electron Microscopy. Methods in Molecular Biology, vol 1117. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-776-1_13

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  • DOI: https://doi.org/10.1007/978-1-62703-776-1_13

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-775-4

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