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.
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References
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.
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.
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.
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.
Douzou, P. (1977) Enzymology at sub-zero temperatures. Adv. Enzymol. 45, 157–272.
Fink, A. L. and Ahmed., I. A. (1976) Formation of stable crystalline enzyme-substrate intermediates at subzero temperatures. Nature 263(5575), 294–297.
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.
Horowitz, R. A. and Woodcock, C. L. (1992) Alternative staining methods for Lowicryl sections. J. Histochem. Cytochem. 40, 123–133.
Millonig, G. (1961) A modified procedure for lead staining of thin sections. J. Biophys. Biochem. Cytol. 11, 736–739.
Newman, G. R. and Hobot, J. A. (1993) Resin Microscopy and On-Section Immunocytochemistry (Springer Lab., ed.) Springer-Verlag, Berlin, Germany.
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.
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.
Villiger, W. (1991) Lowicryl resins, in Colloidal Gold: Principles, Methods, and Applications, vol. 3 (Hayat, M. A., ed.), Academic, San Diego, pp. 59–71.
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.
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.
Griffiths G. (1993) Fine Structure Immunocytochemistry, Springer-Verlag, Berlin, Germany.
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.
Bendayan, M. (1995) Colloidal gold post-embedding immunocytochemistry. Prog. Histochem. Cytochem. 29, 1–159.
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.
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Gounon, P. (1999). Low-Temperature Embedding in Acrylic Resins. In: Nasser Hajibagheri, M.A. (eds) Electron Microscopy Methods and Protocols. Methods in Molecular Biology™, vol 117. Humana Press. https://doi.org/10.1385/1-59259-201-5:111
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DOI: https://doi.org/10.1385/1-59259-201-5:111
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