Advertisement

The Application of LR Gold Resin for Immunogold Labeling

  • J. R. Thorpe
Part of the Methods in Molecular Biology™ book series (MIMB, volume 117)

Abstract

The routine preparation of biological samples for transmission electron microscopy (TEM) usually involves a double-fixation in, first, glutaraldehyde and subsequently in osmium tetroxide (OsO4). The specimens are then dehydrated and embedded in a (heat-polymerized) epoxy resin. While this procedure may produce excellent ultrastructural preservation (see Chapter 1), the antigenicity of most tissue proteins is severely affected. Subsequent successful immunolabeling of such sections with a specific antibody, visualized with either protein A (pA)- or secondary antibody (IgG)-bound gold probe, is thus rare. This loss of antigenicity may reflect denaturation (through fixation, exposure to alcohol, or to the heat of resin polymerization) or inaccessibility (because of high-density crosslinkage of proteins with the fixatives and resin). Protein integrity may be maintained to a degree by adopting a more minimal fixation regime (e.g., omission of OsO4 [see Note 1], decreased concentration of aldehyde and lowered temperature). Additionally, certain antigens in epoxy resin-embedded specimens may also be “unmasked” by “etching” of the sections (e.g., in saturated sodium metaperiodate). However, the remaining component of heat denaturation may still preclude (or hinder; see Note 1) successful immunolocalizations.

Keywords

Toxicity Data Skin Contact Donor Species Preparative Procedure Sodium Metaperiodate 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Thorpe, J. R., Ray, K. P., and Wallis, M. (1990) Occurrence of rare somatomammotrophs in ovine anterior pituitary tissue studied by immunogold labelling and electron microscopy. J. Endocrinol. 124, 67–73.PubMedCrossRefGoogle Scholar
  2. 2.
    Thorpe, J. R. and Wallis, M. (1991) Immunocytochemical and morphometric investigations of mammotrophs, somatotrophs and somatomammotrophs in sheep pituitary cell cultures. J. Endocrinol. 129, 417–422.PubMedCrossRefGoogle Scholar
  3. 3.
    Khawaja, X. Z., Green, I. C., Thorpe, J. R., and Bailey, C. J. (1990) Increased sensitivity to insulin-releasing and glucoregulatory effects of dynorphin A (1-13) and U50,488h on insulin release and glucose homeostasis in genetically obese (ob/ob) mice. Diabetes 39, 1289–1297.PubMedCrossRefGoogle Scholar
  4. 4.
    Khawaja, X. Z., Green, I. C., Thorpe, J. R., and Titheradge, M. A. (1990) The occurrence and receptor specificity of endogenous opioid peptides within the pancreas and liver of the rat-comparison with brain. Biochem. J. 267, 233–240.PubMedGoogle Scholar
  5. 5.
    Thompson, K. S. J., Baines, R. A., Rayne, R. C., Thorpe, J. R., Alef, A., and Bacon, J. P. (1993) Locust VPLI neurons contain three vasopressin-related peptides; VPLI activity reduces cyclic AMP levels in the CNS. Soc. Neurosci. Abstr. 19, 1274.Google Scholar
  6. 6.
    Moore, A. L., Walters, A. J., Thorpe, J. R. Fricaud, A.-C., and Watts, F. Z. (1992) Schizosaccharomyces pombe mitochondria: Morphological, respiratory and protein import characteristics. Yeast 8, 923–933.PubMedCrossRefGoogle Scholar
  7. 7.
    Tobin, A. K., Thorpe, J. R., Hylton, C. M., and Rawsthorne, S. (1989) Spatial and temporal influences on the cell-specific localization of glycine decarboxylase in leaves of wheat (Triticum aestivum) and pea (Pisum sativum). Plant Physiol. 91, 1219–1225.PubMedCrossRefGoogle Scholar
  8. 8.
    Tobin, A. K., Thorpe, J. R., Day, D. A., Wiskitch, J. T., and Moore, A. L. (1990) Immunogold localization of glycine decarboxylase in isolated pea leaf mitochondria (7th. Congress of the Federation of European Societies of Plant Physiology). Physiologia Plantarum 79, Abstract 414.Google Scholar
  9. 9.
    Van Leeuwen, F. (1982) Specific immunocytochemical localizations of neuropep-tides: AUtopian goal?, in Techniques in Immunocytochemistry, vol. 1 (Bullock, G. R. and Petrusz, P., eds.), pp. 283–299.Google Scholar
  10. 10.
    Thorpe, J. R. (1992) A novel methodology for double protein A-gold immunolabeling utilizing the monovalent fragment of protein A. J. Histochem. Cytochem. 40, 435–441.PubMedGoogle Scholar
  11. 11.
    Biberfeld, B., Ghetie, V., and Sjoquist, J. (1975) Demonstration and assaying of IgG antibodies in tissues and on cells by labelled Staphylococcal protein A. J. Immunol. Meth. 6, 249–259.CrossRefGoogle Scholar
  12. 12.
    Goudswaard, J., van der Donk, J. A., Noordzig, A., van Dam, R. H., and Vaerman, J. P. (1978) Protein A reactivity of various mammalian immunoglobulins. Scand. J. Immunol. 8, 21–28.PubMedCrossRefGoogle Scholar
  13. 13.
    Goding, J. W. (1978) Use of Staphylococcal protein A as an immunological reagent. J. Immunol. Meth. 20, 241–253.CrossRefGoogle Scholar
  14. 14.
    Bjorck, L. and Kronvall, G. (1984) Purification and some properties of streptococcal protein G, a novel IgG-binding reagent. J. Immunol. 133, 969–974.PubMedGoogle Scholar
  15. 15.
    McPhail, G. D., Finn, T., and Isaacson, P. G. (1987) A useful low temperature method for post-embedding electron immunocytochemistry in routine histopathology. J. Pathol. 151, 231–238.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 1999

Authors and Affiliations

  • J. R. Thorpe
    • 1
  1. 1.EM and FACS Laboratory, Biological SciencesUniversity of SussexFalmerUK

Personalised recommendations