Abstract
Standard methods for the ultrastructural detection of lipase and sphingomyelinase activities in the skin result in considerable loss of structural preservation, often interfering with accurate delineation of enzyme localization in association with specific organelles. Moreover, poor preservation occurs, even after extensive aldehyde prefixation, owing to the prolonged incubation times needed to detect residual enzyme activity, which often require non- physiological conditions. A modified incubation protocol is described here, which uses microwave irradiation in conjunction with drastically shortened incubation times, resulting in both superior ultrastructural preservation and excellent localization in mammalian epidermis. This method should be useful generally not only for the study of lipase localization in skin, but also in conjuction with the cytochemical detection of a variety of enzymes in various types of tissue.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Elias, P.M., Menon, G.K., Grayson, S. & Brown, B.E. (1988) Membrane structural alterations in murine stratum corneum. Relationship to the localization of polar lipids and phospholipases. J. Invest. Dermatol. 91, 3-10.
Gomori, G. (1948) Histochemical differentiation between esterases. Proc. Soc. Exp. Biol. 67, 4-6.
Hayat, M.A. (1993) Stains and Cytochemical Methods. New York: Plenum Press.
Hochuli, E., Kupfer, E., Maurer, R., Meister, W., Mercadal, Y. & Schmidt, K. (1987) Lipstatin, an inhibitor of pancreatic lipase, produced by Streptomyces toxytricini. II. Chemistry and structure elucidation. J. Antibiot. 40, 1086-91.
Hopwood, D., Yeaman, G. & Milne, G. (1988) Differentiating the effects of microwave and heat on tissue proteins and their crosslinking by formaldehyde. Histochem. J. 20, 341-6.
Jensen, R.G. (1983) Detection and determination of lipase (acylglycerol hydrolase) activity from various sources. Lipids 18, 650-57.
Leonard, J.B. & Shepardson, S.P. (1994) A comparison of heating modes in rapid fixation techniques for electron microscopy. J. Histochem. Cytochem. 42, 383-91.
Login, G.R. & Dvorak, A.M. (1985) Microwave energy fixation for electron microscopy. Am. J. Pathol. 120, 230-43.
Menon, G.K., Grayson, S. & Elias, P.M. (1986) Cytochemical and biochemical localization of lipase and sphingomyelinase activity in mammalian epidermis. J. Invest. Dermatol. 86, 591-97.
Menon, G.K., Hou, E.S.Y. & Elias, P.M. (1991) Avian permeability barrier function reflects mode of sequestration and organization of stratum corneum lipids: Re-evaluation utilizing ruthenium tetroxide staining and lipase cytochemistry. Tissue Cell 48, 445-56.
Menon, G.K., Williams, M.L., Ghadially, R. & Elias, P.M. (1992) Lamellar bodies as delivery systems of hydrolytic enzymes: implications for normal cohesion and abnormal desquamation. Br. J. Dermatol. 126, 337-45.
Mizuhira, V., Notoya, M. & Hasegawa, H. (1990) New tissue fixation for cytochemistry using microwave irradiation. Centered to enzyme cytochemistry, autoradiography, and X-ray microanalysis. J. Histochem. Cytochem. 38, 1039A.
Nagata, T. (1972) Lipase. In Electron Microscopy of Enzymes: Principles and Methods (edited by Hayat, M.A.), Vol. 2, pp. 132-48. New York: Van Nostrand-Reinhold.
Pearse, A.G. (1972) Histochemistry: Theoretical and Applied. London: Longman.
Shri, S.-R., Key, M.E. & Kalra, K.L. (1991) Antigen retrieval in formalin-fixed paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J. Histochem. Cytochem. 39, 741-8.
Tsujita, T., Shirai, K., Saito, Y. & Okuda, H. (1990) Isozymes: Structure, Function, and use in Biology and Medicine, p. 915. New York: Wiley-Liss.
Vethamany, V.G. (1972) Histochemical technique for detecting sphingomyelinase. In Proceedings of 4th International Congress of Histochem. Cytochem. (edited by Takeuchi, T., Ogawa, K. & Fugita, S.), pp. 137-8. Kyoto, Japan.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Rassner, U.A., Crumrine, D.A., Nau, P. et al. Microwave incubation improves lipolytic enzyme preservation for ultrastr uctural cytochemistry. J Mol Hist 29, 387–392 (1997). https://doi.org/10.1023/A:1026438917856
Issue Date:
DOI: https://doi.org/10.1023/A:1026438917856