Skip to main content
SpringerLink
Log in

The influence of embedding media and fixation on the post-embedment ultrastructural demonstration of complex carbohydrates. I. Morphology and periodic acid-thiocarbohydrazide-silver proteinate staining of vicinal diols

  • Papers
  • Published:
The Histochemical Journal Aims and scope Submit manuscript

Summary

The influence of fixation and embedding medium on the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining reactivity in the mouse intestine was studied. It was found that the combination of osmium tetroxide and epoxy resins was the least sensitive for the demonstration of complex carbohydrate with the PA-TCH-SP method. Post-osmication reduced, but did not abolish, PA-TCH-SP reactivity (except for the Golgi complex) when non-epoxy resins were used. The staining pattern of a particular organelle differed depending on the embedding medium used. Golgi cisternae exhibited the most intense PA-TCH-SP reactivity in non-osmicated tissues embedded in non-epoxy resins. Post-osmication of tissues was required to reveal the fine structure of the glycocalyx as well as to preserve the fine structure of tissues embedded in styrene-methacrylate and styrene-Rigolac 2004. The choice of fixation procedures and embedding media in a given study should be governed primarily by the sites of interest.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Adams, C. W. M. (1960) Osmium tetroxide and the Marchi method: reactions with polar and non-polar lipids, protein and polysaccharide.J. Histochem. Cytochem. 8, 262–7.

    Google Scholar 

  • Bahr, C. F. (1954) Osmium tetroxide and ruthenium tetroxide and their reactions with biologically important substances.Expl Cell. Res. 7, 457–79.

    Google Scholar 

  • Barsotti, P. &Marinozzi, V. (1980) PTA staining of osmium-fixed, GMA-embedded tissues. A simple method for revealing the fine structure of polysaccharides and glycoproteins.J. submicrosc. Cytol. 12, 315–9.

    Google Scholar 

  • Borysenko, J. X. &Woods, W. (1979) Density, distribution and mobility of surface anions on a normal/transformed cell pair.Expl Cell. Res. 118, 215–27.

    Google Scholar 

  • Coghill, S. B. &Hopwood, D. (1977) Electron histochemistry of mucosubstances in normal human rectal epithelium.Histochem. J. 9, 231–40.

    Google Scholar 

  • Courtoy, R. &Simar, L. J. (1974) Importance of controls for the demonstration of carbohydrates in electron microscopy with the silver methenamine or the thiocarbohydrazide-silver proteinate methods.J. Microsc. 100, 199–211.

    Google Scholar 

  • Erlandsen, S. L., Parsons, J. A. &Rodning, C. B. (1979) Technical parameters of immunostaining of osmicated tissues in Epoxy sections.J. Histochem. Cytochem. 27, 1286–9.

    Google Scholar 

  • Estes L. W. &Apicella, J. V. (1969) A rapid embedding technique for electron microscopy.Lab. Invest. 20, 159–73.

    Google Scholar 

  • Francioni, G. &Borgioli, G. (1979) Polystyrene embedding: A new method for light and electron microscopy.Stain Technol. 54, 167–72.

    Google Scholar 

  • Geuze, J. J. &Slot, J. W. (1978) Synthesis and secretion of glycoprotein in rat bulbo-urethral (Cowper's) glands. II. Modes of mucus secretion after stimulation by copulation.Am. J. Anat. 152, 391–418.

    Google Scholar 

  • Grinnell, F., Tobleman, M. Q. &Hackenborck, C. R. (1975) The distribution and mobility of anionic sites on the surfaces of baby hamster kidney cells.J. Cell Biol. 66, 470–9.

    Google Scholar 

  • Hayat, M. A. (1970)Principles and Techniques of Electron Microscopy. Biological Applications, Vol. 1. New York, Toronto, London: Van Nostrand Reinhold.

    Google Scholar 

  • Hirsch, J. M. &Fedorko, M. E. (1968) Ultrastructure of human leukocytes after simultaneous fixation with glutaraldehyde and OsO4 and ‘postfixation’ in uranyl acetate.J. Cell Biol. 38, 615–27.

    Google Scholar 

  • Hopwood, D., Logan, K. R. &Milne, G. (1977) Mucosubstances in the normal human eosophageal epithelium. A comparison of periodic acid-silver and phosphotungstic acid techniques.Histochemistry 54, 67–74.

    Google Scholar 

  • Horobin, R. W. &Hague, A. (1971) Periodic acid-Schiff-phosphotungstic acid as an electron histochemical method for ultrathin sections.J. Microsc. 94, 265–71.

    Google Scholar 

  • Hoshino, Y., Shannon, A. &Seligman, A. M. (1976) Comparative study on methods for the ultrastructural demonstration of periodic acid-reactive carbohydrate macromolecules.Acta histochem. cytochem. 9, 255–66.

    Google Scholar 

  • Ito, S. (1974) Form and function of the glycocalyx on free cell surfaces.Phil. Trans. R. Soc. Lond., Ser. B. 268, 55–66.

    Google Scholar 

  • Ito, S. &Karnovsky, M. J. (1968) Formaldehyde-glutaraldehyde fixatives containing trinitro-compounds.J. Cell Biol. 39 168a.

    Google Scholar 

  • Knecht, E. &Hernandez, J. (1978) Ultrastructural localization of polysaccharides in the vacuolar system of an established cell line.Cell Tiss. Res. 193, 473–89.

    Google Scholar 

  • Korn, E. D. (1966) II. Synthesis of bis(methyl 9, 10-dihydroxystearate) osmate from methyl oleate and osmium tetroxide under conditions used for fixation of biological material.Biochim biophys. Acta 116, 317–24.

    Google Scholar 

  • Korn, E. D. (1967) A chromatographic and spectrophotometric study of the products of the reaction of osmium tetroxide with unsaturated lipids.J. Cell Biol. 34, 627–38.

    Google Scholar 

  • Kushida, H. (1960) A new polyester embedding method for ultrathin sectioning.J. Electron Microsc. 9, 113–6.

    Google Scholar 

  • Kushida, H. (1961) A styrene-methacrylate resin embedding method for ultrathin sectioning.J. Electron Microsc. 10, 16–9.

    Google Scholar 

  • La Mont, J. T., Weiser, M. M. &Isselbacher, K. J. (1974) Cell surface glycosyltransferase activity in normal and neoplastic intestinal epithelium of the rat.Cancer Res. 34, 3225–8.

    Google Scholar 

  • Lefranc, G., Chung, Y. T., Barriere, P. &Pradal, G. (1980) Application of the thiocarbohydrazide method for vicinal glycol group detection to the study of gastric mucosa endocrine cells.Histochemistry 66, 277–84.

    Google Scholar 

  • Luciano, L., Reale, E. &Von Engelhardt, W. (1980) The fine structure of the stomach mucosa of the llama (Llama guanocoe). II. The fundic region of the hind stomach.Cell Tiss. Res. 208, 207–28.

    Google Scholar 

  • Luft, J. H. (1961) Improvements in epoxy resin embedding methods.J. Biophys. biochem. Cytol. 9, 409–14.

    Google Scholar 

  • Luft, J. H. (1976) The structure and properties of the cell surface coat.Int. Rev. Cytol. 45, 291–382.

    Google Scholar 

  • Marikovsky, Y., Inbar, M., Danon, D. &Sachs, L. (1974) Distribution of surface charge and concanavalin A-binding sites on normal and malignant transformed cells.Expl Cell Res. 89, 359–67.

    Google Scholar 

  • Marinozzi, V. (1963) The role of fixation in electron staining.J. R. Microsc. Soc. 81, 141–54.

    Google Scholar 

  • Marinozzi, V. (1968) Phosphotungstic acid (PTA) as a stain for polysaccharides and glycoproteins in electron microscopy.Electron Microscopy Proceedings of the 4th European Regional Conference, Rome, Vol. 2, pp. 55–56.

    Google Scholar 

  • Merriam, R. W. (1958) The contribution of lower oxides of osmium to the density of biological specimens in electron microscopy.J. Biophys. biochem. Cytol. 4, 579–82.

    Google Scholar 

  • Milas, N. A., Trepagnier, J. H., Nolan, J. T. &Iliopulos, M. I. (1959) Hydroxylation of olefins and the reaction of osmium tetroxide with 1,2-glycols.J. Am. Chem. Soc. 81, 4730–3.

    Google Scholar 

  • Miller, S. C., Hay, E. D. &Codington, J. F. (1977) Ultrastructural and histochemical differences in cell surface properties of strain-specific and nonstrainspecific TA3 adenocarcinoma cells.J. Cell Biol. 72, 511–29.

    Google Scholar 

  • Mizuhira, V., Shiihashi, M. &Futaesaku, Y. (1981) High-speed electron microscope autoradiographic studies of diffusible compounds.J. Histochem. Cytochem. 29, 143–60.

    Google Scholar 

  • Mohr, W. P. &Cocking, E. C. (1968) A method for preparing highly vacuolated, senescent or damaged plant tissue for ultrastructural study.J. Ultrastruct. Res. 21, 171–81.

    Google Scholar 

  • Morrison, R. T. &Boyd, R. N. (1973)Organic Chemistry, 3rd edn. Boston: Allyn and Bacon.

    Google Scholar 

  • Nakane, P. K. (1971) Application of peroxidase-labeled antibodies to the intracellular localization of hormones.Acta endocr., Suppl.153, 190–204.

    Google Scholar 

  • Nilsson, O., Lindqvist, I. &Ronquist, G. (1973) Decreased surface charge of mouse blastocysts at implantation.Expl Cell Res. 83, 421–3.

    Google Scholar 

  • Pearse, A. G. E. (1975)Histochemistry, Theoretical and Applied. Edinburgh, London: Churchill Livingstone.

    Google Scholar 

  • Pigman, W. (1977) General aspects. InThe Glycoconjugates, Vol. 1, Mammalian Glycoproteins and Glycolipids (edited byHorowitz, M. I. andPigman, W.), pp. 1–14. New York, San Francisco, London: Academic Press.

    Google Scholar 

  • Pratt, S. A. &Napolitano, L. (1969) Osmium binding to the surface coat of intestinal microvilli in the cat under various conditions.Anat. Rec. 165, 197–210.

    Google Scholar 

  • Rambourg, A. (1971) Morphological and histochemical aspects of glycoproteins at the surface of animal cells.Int. Rev. Cytol. 31, 57–114.

    Google Scholar 

  • Reynolds, E. S. (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy.J. Cell Biol. 17, 208–12.

    Google Scholar 

  • Riemersma, J. C. (1968) Osmium tetroxide fixation of lipids for electron microscopy: a possible reaction mechanism.Biochim. biophys. Acta 152, 718–27.

    Google Scholar 

  • Ryter, A. &Kellenberger, E.(1958) L'inclusion au polyester pour l'ultramicrotomie.J. Ultrastruct. Res. 2, 200–14.

    Google Scholar 

  • Sato, A. &Spicer, S. S. (1980) Ultrastructural cytochemistry of complex carbohydrates of gastric epithelium in the guinea pig.Am. J. Anat. 159, 307–29.

    Google Scholar 

  • Shinagawa, Y. &Ogura, M. (1960) Polystyrene embedding method for ultrathin sectioning.J. Electron Microsc. 9, 148–50.

    Google Scholar 

  • Shinagawa, Y. &Uchida, Y. (1961) On the specimen damage of spinal cord due to polymerization of embedding media.J. Electron Micros. 10, 86–91.

    Google Scholar 

  • Silva, M. T. (1967) Electron microscopy study on the effect of oxidation of ultrathin sections ofBacillus cereus andBacillus megaterium.J. Ultrastruct. Res. 18, 345–53.

    Google Scholar 

  • Simson, J. A. V. (1977) The influence of fixation on the carbohydrate cytochemistry of rat salivary gland secretory granules.Histochem. J. 9, 645–57.

    Google Scholar 

  • Skutelsky, E. &Danon, D. (1969) Reduction in surface charge as an explanation of the recognition by macrophages of nuclei expelled from normoblasts.J. Cell Biol. 43, 8–15.

    Google Scholar 

  • Skutelsky, E. &Farquhar, M. G. (1976) Variations in distribution of con A receptor sites and aniomic groups during red blood cell differentiation in the rat.J. Cell Biol. 71, 218–31.

    Google Scholar 

  • Spicer, S. S., Mochizuki, I., Setser, M. E. &Martinez, J. R. (1980) Complex carbohydrates of rat tracheobronchial surface epithelium visualized ultrastructurally.Am. J. Anat. 158, 93–109.

    Google Scholar 

  • Spicer, S. S. &Schulte, B. A. (1982) Ultrastructural methods for localizing complex carbohydrates.Human Path. 13, 343–54.

    Google Scholar 

  • Spiro, R. G. (1969) Glycoproteins: Their biochemistry, biology and role in human disease.New Engl J. Med. 281, 991–1001.

    Google Scholar 

  • Spurr, A. R. (1969) A low viscosity epoxy resin embedding medium for electron microscopy.J. Ultrastruct. Res. 26, 31–43.

    Google Scholar 

  • Takamiya, H., Batsford, S. &Vogt, A. (1980) An approach to post-embedding staining of protein (immunoglobulin) antigen embedded in plastic: Prerequisites and limitations.J. Histochem. Cytochem. 28, 1041–9.

    Google Scholar 

  • Thiery, J. P. (1967) Mise en evidence des polysaccharides sur coupes fines en microscopie electronique.J. Microscopie 6, 987–1018.

    Google Scholar 

  • Tixier-Vidal, A. &Picart, R. (1971) Electron microscopic localization of glycoproteins in pituitary cells of duck and quail.J. Histochem. Cytochem. 19, 775–97.

    Google Scholar 

  • Wattel, W., Geuze, J. J. &De Rooij, D. G. (1977a) Ultrastructural and carbohydrate histochemical studies on the differentiation and renewal of mucous cells in the rat gastric fundus.Cell Tiss. Res. 176, 445–62.

    Google Scholar 

  • Wattel, W., Geuze, J. J., De Rooij, D. G. &Davids, J. A. G. (1979b) Renewal of mouse gastric mucous cells following fast neutron irradiation. An ultrastructural and carbohydrate histochemical study.Cell Tiss. Res. 183, 303–18.

    Google Scholar 

  • Weiser, M. M. (1973) Intestinal epithelial cell surface membrane glycoprotein synthesis. I. An indicator of cellular differentiation.J. biol. Chem. 248, 2536–41.

    Google Scholar 

  • 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. 25, 947–60.

    Google Scholar 

  • Yamada, K. &Hoshino, M. (1976) The reactions of rabbit gall bladder epithelium to electron microscopic staining for carbohydrates with 1,2-glycol groupings.Acta histochem. 56, 156–69.

    Google Scholar 

  • Yamamoto, N. &Yasuda, K. (1977) Use of a water soluble carbodiimide as a fixing reagent.Acta histochem. cytochem. 10, 14–37.

    Google Scholar 

  • Yanagimachi, R., Noda, Y. D., Fujimoto, M. &Nicolson, G. L. (1972) The distribution of negative surface charges on mammalian spermatozoa.Am. J. Anat. 135, 497–520.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Pathology, Medical University of South Carolina, 29425, Charleston, South Carolina, U.S.A.

    G. N. Thomopoulos, B. A. Schulte & S. S. Spicer

Authors
  1. G. N. Thomopoulos
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. B. A. Schulte
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. S. S. Spicer
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Thomopoulos, G.N., Schulte, B.A. & Spicer, S.S. The influence of embedding media and fixation on the post-embedment ultrastructural demonstration of complex carbohydrates. I. Morphology and periodic acid-thiocarbohydrazide-silver proteinate staining of vicinal diols. Histochem J 15, 763–784 (1983). https://doi.org/10.1007/BF01003340

Download citation

  • Received:

  • Revised:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01003340

Keywords

Search

Navigation