Specimen Preparation for Immunocytochemistry

  • Gillian Brown
  • Anthony Wong
  • Julian E. Beesley

Abstract

Specimen preparation for immunolabeling should stabilize the tissue architecture and expose the antigen for immunolabeling. Techniques vary from simply flooding a culture well with fixative followed by rinsing and immunolabeling the specimen to sophisticated surgical techniques, complex specimen freezing, substitution, embedding, and sectioning. Naturally, any perturbation of the structure of the tissue at this stage will be apparent when defining the immunolabeling. Dislocation of tissue components will give false localization and, at worst, the specimen details will not be interpretable. Specimen preparation involves fixation, a complex chemical and physical process, to stabilize the specimen and further manipulations to prepare the specimen for immunolabeling. Fixation was described in Chapter 2; the remaining manipulations will be described here.

Keywords

Formalin Acetone Agar Mold Epoxy 

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References

  1. Al-Hazza, A. A., and Bowen, I. D. 1998. Improved cytochemical methods for demonstrating cell death using LR White as an embedding medium. Histochemical Journal 30:897–902.CrossRefGoogle Scholar
  2. Anderson, G., and Gordon, K. C., 1996. Tissue processing, microtomy and paraffin sections. In J. D. Bancroft and A. Stevens, eds., Theory and Practice of Histological Techniques (47–80). New York: Churchill Livingstone.Google Scholar
  3. Bancroft, J., and Palmer, J., 1996. Frozen and related sections. In J. D. Bancroft and A. Stevens, eds., Theory and Practice of Histological Techniques (69–80). New York: Churchill LivingstoneGoogle Scholar
  4. Beesley, J. E., Day, S. E., Betts, M. P., and Thorley, C. M. 1984. Immunocytochemical labelling of Bacteroides nodosus pili using an immunogold technique. Journal of General Microscopy 130(6):1481–1487.Google Scholar
  5. Brorson, S. H. 1998. Antigen detection on resin sections and methods for improving the immunogold labeling by manipulating the resin. Histology and Histopathology 13(1):275–281.PubMedGoogle Scholar
  6. Carlemalm, E., Villiger, W., Hobot, J. A., Acetarin, J.-D. and Kellenberger. 1985. Low temperature embedding with Lowicryl resins: two new formulations and some applications. Journal of Microscopy 140(1):55–63.PubMedCrossRefGoogle Scholar
  7. Causton, B. E. 1984. The choice of resins for electron immunocytochemistry. In J. M. Polak and I. A. Varndell, eds., Immunolabelling for Electron-Microscopy (29–36). Amsterdam, New York: Elsevier Science Publishers B. V.Google Scholar
  8. Da Silva, P. P., Kachar, B., Torrisi, M. R., Brown, C., and Parkinson, C. 1981a. Freeze-fracture cytochemistry: replicas of critical point-dried cells and tissues after fracture label. Science. 213(4504):230–233.CrossRefGoogle Scholar
  9. Da Silva, P. P., Parkinson, C., and Dwyer, N. 1981b. Thin sections and tissues after la-belling of fracture faces. Journal of Histochemistry and Cytochemistry 29(8):917–928.CrossRefGoogle Scholar
  10. Da Silva, P. P., and Kan, F. W. K. 1984. Label-Fracture: A method for high resolution labelling of cell surfaces. The Journal of Cell Biology. 99:1156–1161CrossRefGoogle Scholar
  11. Doane, F. W., and Anderson, N. 1987. Methods for preparing specimens for electron microscopy. In F. W. Doane and N. Anderson, eds., Electron Microscopy in Diagnostic Virology (15–17). Cambridge University Press.Google Scholar
  12. Dudek, R. W., Varndell, I. M., and Polak, J. M. 1984. Combined quick-freeze and freeze-drying techniques for improved electron immunocytochemistry. In J. M. Polak and I. A. Varndell, eds., Immunolabelling for Electron-Microscopy (235–248). Amsterdam, New York: Elsevier Science Publishers B. V.Google Scholar
  13. Fujikawa, S. 1991. Freeze-fracture techniques. In J. R. Harris, ed. Electron Microscopy in Biology: A Practical Approach (17–38). Oxford, UK: IRL Press at Oxford University Press.Google Scholar
  14. Fujimoto, K. 1995. Freeze-fracture replica electron microscopy combined with SDS digestion for cytochemical labeling of integral membrane proteins. Journal of Cell Science 108:3443–3449.PubMedGoogle Scholar
  15. Fujimoto, K., Umaeda, M., and Fujimoto, T. 1996. Transmembrane phospolipid distribution revealed by freeze-fracture replica labeling. Journal of Cell Science 109:2453–2460.PubMedGoogle Scholar
  16. Germain, F., and Stevens, A., 1996. Resin embedding media. In J. D. Bancroft and A. Stevens, eds., Theory and Practice of Histological Techniques (555–567). New York: Churchill LivingstoneGoogle Scholar
  17. Griffiths, G., McDowall, A., Back, R., and Dubochet, J. 1984 On the preparation of cryo-sections for immunocytochemistry. Journal of Ultrastructural Research 89:65–78.CrossRefGoogle Scholar
  18. Hirsch, J. G., and Fedorko, M. E. 1968. Ultrastructure of human leucocytes after simultaneous fixation with glutaraldehyde and osmium tetroxide and ‘pot fixation’ in uranyl acetate. Journal of Cell Biology 38:615–27.PubMedCrossRefGoogle Scholar
  19. Hobot, J. A., and Newman, R. 1996. Immunomicroscopy: resin techniques and on-section labelling with immunocolloidal gold or immunoperoxidase-planning a protocol. Scanning Microscopy 10(1):121–145.PubMedGoogle Scholar
  20. Hodges, G. M., Southgate, J, and Toulon, E. C. 1987. Colloidal gold-a powerful tool in scanning electron microscope immunocytochemistry: an overview of bioapplications. Scanning Microscopy 1(1):301–318.PubMedGoogle Scholar
  21. Hunter, E. E. 1993. Practical Electron Microscopy: A Beginner’s Illustrated Guide, 2nd ed. Cambridge, New York, Victoria: Cambridge University Press.CrossRefGoogle Scholar
  22. Hyatt, A. D., Eaton, B. T., and Lunt, R. 1987. The grid-cell-culture technique: the direct examination of virus infected cells and progeny viruses. Journal of Microscopy 145(1):97–106.PubMedCrossRefGoogle Scholar
  23. Kuemmel, T. A., Thiele, J., Varus, E., Hafenrichter, E. G., Suedkamp, M., and Fischer, R. 1996. An improved postembedding technique for ultrastructural studies of lectin. Journal of Submicroscopical Cytology and Pathology 28(2):197–208.Google Scholar
  24. Linner, J. G., Livesey, S. A., Harrison, D. S., and Steiner, A. L. 1986. A new technique for removal of amorphous phase tissue water without ice crystal damage: a preparative method for ultrastructural analysis and immunoelectron microscopy. Journal of Histochemistry and Cytochemistry 34(9):1123–1135.PubMedCrossRefGoogle Scholar
  25. Luft, J. H. 1961. Improvements in epoxy resin embedding methods. Journal of Biophysical and Biochemical Cytology 9:409–414.PubMedCrossRefGoogle Scholar
  26. Maxwell, D. J., Todd, A. J., and Kerr, R. 1995. Colocalization of glycine and GABA in synapses on spinomedullary neurons. Brain Research 690(1):127–132.PubMedCrossRefGoogle Scholar
  27. Monaghan, P., Perusinghe, N., and Muller, M. 1998. High-pressure freezing for immunocytochemistry. Journal of Microscopy 192(3):248–258.PubMedCrossRefGoogle Scholar
  28. Morse, A. 1996, Diagnostic cytopathology; specimen collection and preparation. In J. D. Bancroft and A. Stevens, eds., Theory and Practice of Histological Techniques (513–554). New York: Churchill Livingstone.Google Scholar
  29. Neuhaus, E. M., Horstmann, H., Almers, W., Maniak, M., and Soldati, T. 1998. Ethanefreezing/methanol-fixation of cell monolayers: a procedure for improved preservation of structure and antigenicity for light and electron microscopies. Journal of Structural Biology 121:326–342.PubMedCrossRefGoogle Scholar
  30. Pegg-Feige, K., and Doane, E W. 1983. Effect of specimen support film in solid phase immunoelectron microscopy. Journal of Virological Methods 7(5–6):315–319.PubMedCrossRefGoogle Scholar
  31. Preda, P., Pileri, S., and Pasquinelli, G., 1995. Microwave oven heating enhances the ultrastructural detection of antigens in Bioacryl-embedded tissue sections. Microscopy Research and Technique 32(1):75–76.PubMedCrossRefGoogle Scholar
  32. Priestly, J. V. 1984. Pre-embedding ultrastructural immunocytochemistry: immunoenzyme techniques. In J. M. Polak and I. A. Varndell, eds., Immunolabelling for Electron-Microscopy (37–52). Amsterdam, New York: Elsevier Science Publishers B.V.Google Scholar
  33. Robards, A. W., and Sleytr, U. B. 1985. Low Temperature Methods in Biological Electron Microscopy. Amsterdam: Elsevier Science Publishers, B.V.Google Scholar
  34. Rothe, F., Brosz, M., and Storm-Mathisen, J. 1995. Quantitative ultrastructural localization of glutamate dehydrogenase in rat cerebellar cortex. Neuroscience 64(4):iii-xvi.PubMedCrossRefGoogle Scholar
  35. Smith, M., and Croft, S. 1991. Embedding and thin section preparation. In J. R. Harris, ed., Electron Microscopy in Biology: A Practical Approach (17–38) Oxford, UK: IRL Press at Oxford University Press.Google Scholar
  36. Springall, D. R. 1986. Immunocytochemistry in Diagnostic Cytology. In J. M. Polak and S. van Noorden, eds., Immunocytochemistry Modern Methods and Applications (547–567). Bristol: John Wright & Son, Ltd.Google Scholar
  37. Studer, D., Michel, M., Wohlwend, M., Hunziker, E. B., and Buschmann, M. D. 1995. Vitrification of articular cartilage by high-pressure freezing. Journal of Microscopy 179(3):321–332.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2001

Authors and Affiliations

  • Gillian Brown
  • Anthony Wong
  • Julian E. Beesley

There are no affiliations available

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