Advertisement

Low-Temperature Embedding in Acrylic Resins

  • Pierre Gounon
Part of the Methods in Molecular Biology™ book series (MIMB, volume 117)

Abstract

It has been demonstrated that enzymes (5,6) can maintain their structure and their activity at very low temperature in concentrated organic solvent. Therefore, in order to minimize molecular thermal vibration, which can have adverse effects on specimens weakly fixed with paraformaldehyde, one can dehydrate samples partially or totally at low temperature. Carlemalm et al., 1982 (3) introduced the PLT technique (progressive lowering of temperature) that combines increasing organic solvent concentration with decreasing temperature, after which infiltration and polymerization are carried out. The results obtained with Lowicryls clearly show the advantages of this approach to obtain good structural preservation of cellular contents and ultrastructure. Furthermore, the PLT method employs low temperature to reduce protein denaturation and to maintain a degree of hydration, which may be important in preserving protein structural conformation. Specimens suffer most during dehydration by organic solvents, mainly ethanol, whereas final infiltration by resin monomers and polymerization seems to be less critical.

Keywords

Acrylic Resin Aqueous Uranyl Acetate Neat Resin Pure Resin Resin Monomer 
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.
    Benichou, J. C., Frehel, C., and Ryter, A. (1990) Improved sectioning and ultrastructure of bacteria and animal cells embedded in Lowicryl. J. Electron Microsc. Tech. 14, 289–97.PubMedCrossRefGoogle Scholar
  2. 2.
    Bogers, J. J., Nibbeling, H. A., Deelder, A. M., and Van Marck, E. A. (1996) Quantitative and morphological aspects of Unicryl versus Lowicryl K4M embedding in immunoelectron microscopic studies. J. Histochem. Cytochem. 44, 43–48.PubMedGoogle Scholar
  3. 3.
    Carlemalm, E., Garavito, R. M., and Villiger, W. (1982) Resin development for electron microscopy and an analysis of embedding at low temperature. J. Microsc. (Oxford) 126, 123–143.Google Scholar
  4. 4.
    Chevalier, J., Yi, J., Michel, O., and Tang, X. M. (1997) Biotin and digoxigenin as labels for light and electron microscopy in situ hybridization probes: Where do we stand? J. Histochem. Cytochem. 45, 481–491.PubMedGoogle Scholar
  5. 5.
    Douzou, P. (1977) Enzymology at sub-zero temperatures. Adv. Enzymol. 45, 157–272.PubMedGoogle Scholar
  6. 6.
    Fink, A. L. and Ahmed., I. A. (1976) Formation of stable crystalline enzyme-substrate intermediates at subzero temperatures. Nature 263(5575), 294–297.PubMedCrossRefGoogle Scholar
  7. 7.
    Goping, G., Kuijpers, G. A. J., Vinet, R., and Pollard, H. B. (1996) Comparison of LR White and Unicryl as embedding media for light and electron immunomicroscopy of chromaffin cells. J. Histochem. Cytochem. 44, 289–295.PubMedGoogle Scholar
  8. 8.
    Horowitz, R. A. and Woodcock, C. L. (1992) Alternative staining methods for Lowicryl sections. J. Histochem. Cytochem. 40, 123–133.PubMedGoogle Scholar
  9. 9.
    Millonig, G. (1961) A modified procedure for lead staining of thin sections. J. Biophys. Biochem. Cytol. 11, 736–739.PubMedCrossRefGoogle Scholar
  10. 10.
    Newman, G. R. and Hobot, J. A. (1993) Resin Microscopy and On-Section Immunocytochemistry (Springer Lab., ed.) Springer-Verlag, Berlin, Germany.Google Scholar
  11. 11.
    Scala, C., Cenacchi, G., Ferrari, C., Pasqualini, G., Preda, P., and Manara, G. C. (1992) A new acrylic resin formulation: A useful tool for histological, ultrastructural, and immunocytochemical investigations. J. Histochem. Cytochem. 40, 1799–1804.PubMedGoogle Scholar
  12. 12.
    Scala, C., Preda, P., Cenacchi, G., Martinelli, G. N., Manara, G. C., and Pasquinelli, G. (1993) Anew polychrome stain and simultaneous methods of histological, his 124 tochemical and immunohistochemical stainings performed on semithin sections of Bioacryl-embedded human tissues. Histochem. J. 25, 670–677.PubMedCrossRefGoogle Scholar
  13. 13.
    Villiger, W. (1991) Lowicryl resins, in Colloidal Gold: Principles, Methods, and Applications, vol. 3 (Hayat, M. A., ed.), Academic, San Diego, pp. 59–71.Google Scholar
  14. 14.
    Weibull, C., Villiger, W., and Carlemalm, E. (1984) Extraction of lipids during freeze-substitution of Acholeplasma laidlawii cells for electron microscopy. J.Microsc. 134, 213–216.PubMedGoogle Scholar
  15. 15.
    Puvion-Dutilleul, F. (1993) Protocol of electron microscope in situ nucleic acid hybridization for the exclusive detection of double-stranded DNA sequences in cells containing large amounts of homologous single-stranded DNA and RNA sequences: Application to adenovirus type 5 infected HeLa cells. Microsc. Res. Tech. 25, 2–11.PubMedCrossRefGoogle Scholar
  16. 16.
    Griffiths G. (1993) Fine Structure Immunocytochemistry, Springer-Verlag, Berlin, Germany.Google Scholar
  17. 17.
    Roth, J., Bendayan, M., Carlemalm, E., Villiger, W., and Garavito, R. M. (1981) Enhancement of structural preservation and immunocytochemical staining in low temperature embedded pancreatic tissue. J. Histochem. Cytochem. 29, 663–671.PubMedGoogle Scholar
  18. 18.
    Bendayan, M. (1995) Colloidal gold post-embedding immunocytochemistry. Prog. Histochem. Cytochem. 29, 1–159.PubMedGoogle Scholar
  19. 19.
    Gounon, P. and Rolland, J.-P. (1998) Modification of Unicryl composition for rapid polymerization at low temperature without alteration of immunocytochemical sensitivity. Micron 29(4), 293–296.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 1999

Authors and Affiliations

  • Pierre Gounon
    • 1
  1. 1.Station Centrale de Microscopie ElectroniqueInstitut PasteurParisFrance

Personalised recommendations