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The Histochemical Journal

, Volume 12, Issue 4, pp 419–434 | Cite as

Electron microscopic immunocytochemical localization of intracellular antigens in cultured cells: The EGS and ferritin bridge procedures

  • Mark C. Willingham
Papers

Summary

A new primary fixative, ethyldimethylaminopropyl carbodi-imide-glutaraldehyde-Tris, has been combined with the use of saponin for membrane permeabilization to yield a procedure which preserves ultrastructural morphology, yet retains a cytoplasmic matrix permeable to globulin molecules. This allows pre-embedding localization of intracellular protein antigens in cultured cells by fluorescence or electron microscopy. A further combination of these methods with the ferratin bridge' technique has allowed discrete localization which is quantifiable. Together, these methods yield an overall technique which provides high quality ultrastructural morphological preservation and precise antigen localization. Examples of the localization of α2-macroglobulin, actin, SV40 T-antigen, tubulin and p60 src are demonstrated. Extension of these methods from cultured cells to intact tissue should be possible without major changes.

Keywords

Electron Microscopy Ferritin Major Change Membrane Permeabilization Saponin 
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.

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References

  1. Ainsworth, S. K. &Karnovsky, M. J. (1972) An ultrastructural staining method for enhancing the size and electron opacity of ferritin in thin sections.J. Histochem. Cytochem. 20, 225–9.Google Scholar
  2. Cabral, F., Willingham, M. C. & Gottesman, M. M. (1980) Ultrastructural localization to 10 nm filaments of an insoluble 58K protein in cultured fibroblasts.J. Histochem. Cytochem. (in press).Google Scholar
  3. Debrabander, M., Demey, J., Joniau, M. &Gevens, S. (1977) Immunocytochemical visualization of microtubules and tubulin at the light and electron-microscopic level.J. Cell. Sci. 28, 283–301.Google Scholar
  4. Demey, J., Hoebeke, J., Debrabander, M., Gevens, G. &Joniau, M (1976) Immunoperoxidase visualization of microtubules and microtubular proteins.Nature, Lond. 264, 273–5.Google Scholar
  5. Ohtsuki, I., Manzi, R. M., Palade, G. E. &Jamieson, J. D. (1978) Entry of macromolecular tracers into cells fixed with low concentrations of aldehydes.Biol. Cell. 31, 119–26.Google Scholar
  6. Reynolds, E. S. (1963) The use of lead citrate at high pH as an electron opaque stain in electron microscopy.J. Cell Biol. 17, 208–13.Google Scholar
  7. Willingham, M. C. &Yamada, S. S (1979) Development of a new primary fixative for electron microscopic immunocytochemical localization of intracellular antigens in cultured cells.J. Histochem. Cytochem. 27, 947–60.Google Scholar
  8. Willingham, M. C., Spicer, S. S. &Graber, C. D. (1971) Immunocytologic labeling of calf and human lymphocyte surface antigens.Lab. Invest. 25, 211–9.Google Scholar
  9. Willingham, M. C., Yamada, S. S. &Pastan, I. (1978) Ultrastructural antibody localization of α2-marcoglobulin in membrane-limited vesicles in cultured cells.Proc. natn. Acad. Sci. U.S.A. 75, 4359–63.Google Scholar
  10. Willingham, M. C., Jay, G. &Pastan, I. (1979) Localization of the avian sarcoma virussrc gene product to the plasma membrane of transformed cells by electron microscopic immunocytochemistry.Cell 18, 125–34.Google Scholar
  11. Willingham, M. C., Yamada, S. S. & Pastan, I. (1980) Ultrastructural localization of tubulin in cultured fibroblastsJ. Histochem. Cytochem. (in press).Google Scholar

Copyright information

© Chapman and Hall Ltd 1980

Authors and Affiliations

  • Mark C. Willingham
    • 1
  1. 1.Laboratory of Molecular BiologyNational Cancer InstituteBethesdaUSA

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