Histochemie

, Volume 19, Issue 1, pp 64–74 | Cite as

Preparation of tissues for localization of sex hormones by autoradiography

I. Loss of radioactive material effected by the use of some conventional methods
  • Arne Attramadal
Article
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Summary

Tissues of rats given 3H-oestradiol were prepared for autoradiography according to methods commonly used in light and electron microscopy.

By formalin fixation large amounts of radioactive material were lost, both in the fixative and during dehydration. Altogether 78.6±7.5 per cent was extracted from uterine tissue, while 49.0±4.6 per cent was lost from liver tissue removed 15 minutes after the injection. Significantly more radioactivity was lost in the fixative from liver tissue than from uterine. In the former fixation accounted for about 60 per cent of the loss, whereas in the latter it was responsible for about 25 per cent.

Osmium tetroxide fixation was found to retain the radioactivity of liver and uterine tissue almost completely. However, large amounts were invariably extracted during dehydration. Although only 3.9±1.2 per cent of the radioactivity of uterine tissue diffused into the fixative, 72.8±12.4 per cent was extracted during ethanol dehydration. A heavy loss was also registered when dehydration and infiltration were carried out in glycol methacrylate.

Glutaraldehyde perfusion and postfixation with osmium tetroxide retained almost completely the radioactivity of uterine and pituitary tissue. Nevertheless, nearly all of it was extracted during ethanol/propylene oxide dehydration and Epon embedding.

The methods studied are not adequate for accurate autoradiographic localization of oestradiol.

Keywords

Glycol Oestradiol Formalin Liver Tissue Methacrylate 
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. Adams, C. W. M.: Histochemistry of lipids. In: Neurohistochemistry C. W. M. Adams, (editor), p. 6–66. Amsterdam-London-New York: Elsevier Publ. Co. 1965.Google Scholar
  2. Attramadal, A.: A method for exact intracellular localization of tritiated sex hormones by autoradiography. Acta path. microbiol. scand. 61, 150–151 (1964).Google Scholar
  3. —: Distribution and site of action of oestradiol in the brain and pituitary gland of the rat following intramuscular administration. In: Proc. 2nd Int. Congr. Endocrin., p. 612–616. Amsterdam: Excerpta Medica Foundation, Int. Congr. Ser. No 83, 1964.Google Scholar
  4. Dallam, B. D.: Determination of protein and lipid lost during osmic acid fixation of tissues and cellular particulates. J. Histochem. Cytochem. 5, 178–181 (1957).Google Scholar
  5. Dulcino, J., R. Bosco, W. G. Verly, and J. B. Maisin: Assay of tritium and carbon-14 in animal tissues by liquid scintillation. Clin. chim. Acta 8, 58–65 (1963).Google Scholar
  6. Eik-Nes, K., J. A. Schellman, R. Lumry, and L. T. Samuels: The binding of steroids to protein. I. Solubility determinations. J. biol. Chem. 206, 411–419 (1954).Google Scholar
  7. Fraenkel-Conrat, H., and D. K. Mecham: The reaction of formaldehyde with proteins. VII. Demonstration of intermolecular crosslinking by means of osmotic pressure measurements. J. biol. Chem. 177, 477–486 (1949).Google Scholar
  8. French, D., and J. T. Edsall: The reactions of formaldehyde with amino acids and proteins. Advanc. Protein Chem. 2, 277–335 (1945).Google Scholar
  9. Giacomelli, F., J. Wiener, and D. Spiro: Cytological alterations related to stimulation of the zona glomerulosa of the adrenal gland. J. Cell Biol. 26, 499–522 (1965).Google Scholar
  10. Hayes, T. L., F. T. Lindgren, and J. W. Gofman: A quantitative determination of the osmium tetroxide-lipoprotein interaction. J. Cell Biol. 19, 251–255 (1963).Google Scholar
  11. Hovig, T., and A. Attramadal: Unpublished.Google Scholar
  12. Jensen, E. V., and H. I. Jacobson: Basic guides to the mechanism of estrogen action. Recent Progr. Hormone Res. 18, 387–414 (1962).Google Scholar
  13. —, E. R. De Sombre, and P. W. Jungblut: Interaction of estrogens with receptor sites in vivo and in vitro. In: Proc. 2. Int. Congr. on Hormonal Steroids L. Martini, F. Fraschini, M. Motta, (editors), p. 492–500. Amsterdam: Excerpta Medica Foundation 1967.Google Scholar
  14. —, T. Suzuki, T. Kawashima, W. E. Stumpf, P. W. Jungblut, and E. R. De Sombre: A two-step mechanism for the interaction of estradiol with rat uterus. Proc. nat. Acad. Sci. (Wash.) 59, 632–638 (1968).Google Scholar
  15. Karlsson, U., and R. L. Schultz: Fixation of the central nervous system for electron microscopy by aldehyde perfusion. Preservation with aldehyde perfusates versus direct perfusion with osmium tetroxide with special reference to membranes and the extra-cellular space. J. Ultrastruct. Res. 12, 160–186 (1965).Google Scholar
  16. King, R. J. B., J. Gordon, and D. R. Inman: The intracellular localization of oestrogen in rat tissues. J. Endocr. 32, 9–15 (1965).Google Scholar
  17. - J. Gordon, and L. Martin: The association of (6.7-3H2)oestradiol with nuclear chromatin. Biochem. J. 97, 28 p., (1965).Google Scholar
  18. Korn, E. D.: Structure of biological membranes. Science 153, 1491–1498 (1966a).Google Scholar
  19. —: Modification of oleic acid during fixation of amoebae by osmium tetroxide. Biochim. biophys. Acta (Amst.) 116, 325–335 (1966b).Google Scholar
  20. —, and R. A. Weisman: I. Loss of lipids during preparation of amoebae for electron microscopy. Biochim. biophys. Acta (Amst.) 116, 309–316 (1966).Google Scholar
  21. Maurer, H. R., and G. R. Chalkley: Some properties of a nuclear binding site of estradiol. J. molec. Biol. 27, 431–441 (1967).Google Scholar
  22. Pearse, A. G. E.: Histochemistry. Theoretical and applied, p. 53–74. London: J. & A. Churchill Ltd. 1960.Google Scholar
  23. Rogers, A. W., and J. F. Moran: Evaluation of quench correction in liquid scintillation counting by internal, automatic external, and channels' ratio standardization methods. Analyt. Biochem. 16, 206–219 (1966).Google Scholar
  24. Roozemond, R. C.: Thin layer chromatographic study of lipid extraction from cryostat sections of rat hypothalamus by some fixatives. J. Histochem. Cytochem. 15, 526–529 (1967).Google Scholar
  25. Ruchelman, M. W.: Solubility studies of estrone in organic solvents using gas-liquid chromatography. Analyt. Biochem. 19, 98–108 (1967).Google Scholar
  26. Sjöblom, L.: Studies on the solubilization of oestrone and its percutaneous absorption in mice. Acta Acad. Aboensis Math. Phys. 20, (No 14) 34–42 (1956).Google Scholar
  27. Snart, R. S.: Physico-chemical bases of hormonal action. In: Proc. 2. Int. Congr. Endocrin., p. 1313–1323. Amsterdam: Excerpta Medica Foundation, Int. Congr. Ser. No. 83, 1964.Google Scholar
  28. Stone, G. M.: The uptake of tritiated oestrogens by various organs of the ovariectomized mouse following subcutaneous administration. J. Endocr. 27, 281–288 (1963).Google Scholar
  29. Stumpf, W. E., and L. J. Roth: High resolution autoradiography with dry mounted, freezedried frozen sections. Comparative study of six methods using two diffusible compounds 3H-estradiol and 3H-mesobilirubinogen. J. Histochem. Cytochem. 14, 274–287 (1966).Google Scholar
  30. Toft, D., and J. Gorski: A receptor molecule for estrogens: Isolation from the rat uterus and preliminary characterization. Proc. nat. Acad. Sci. (Wash.) 55, 1574–1581 (1966).Google Scholar
  31. Wigglesworth, V. B.: The use of osmium in the fixation and staining of tissues. Proc. roy. Soc. B 147, 185–199 (1957).Google Scholar
  32. Wilske, K. R., and R. Ross: Autoradiographic localization of lipid- and water-soluble compounds: A new approach. J. Histochem. Cytochem. 13, 38–43 (1965).Google Scholar

Copyright information

© Springer-Verlag 1969

Authors and Affiliations

  • Arne Attramadal
    • 1
  1. 1.The University Institute of Pathological AnatomyRikshospitaletNorway

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