Summary
The histochemical localization of cholesterol using oxidized diaminobenzidine as the final reaction product was studied at the electron microscopical level and compared with the digitonin method of cholesterol localization based on cholesterol digitonide as the final reaction product. Tissue chopper sections of fixed rat adrenal glands were incubated at 37° C in a medium consisting of 0.8 units/ml cholesterol oxidase, 1.4 units/ml cholesterol ester hydrolase, 50 units/ml horseradish peroxidase, 0.5 mg/ml diaminobenzidine, 0.1% v/v Triton X-100 (or Surfal) and an endogenous peroxidase inhibitor in 0.1m phosphate buffer, pH 7.0. An electron-dense osmiophilic reaction product was observed in many lipid droplets, intracellular vesicles and focally around mitochondria. Appropriate control experiments indicated that deposition of reaction product depended on the presence of cholesterol and the necessary enzymes. Comparison studies using digitonin confirmed the presence of cholesterol in the lipid droplets, but ultrastructural distortion limited the resolution of the more discrete deposits of cholesterol such as around mitochondria. The enzyme method permits finer resolution of these discrete deposits of cholesterol than the digitonin method because it does not cause distortion of cellular ultrastructure attributed to the formation of cholesterol digitonide. The enzyme method or a combination of enzyme and digitonin enables localization of free, esterified or total cholesterol.
Similar content being viewed by others
References
Allain, C., Chan, C., Richmond, W. &Fu, P. (1974) Enzymatic determination of total serum cholesterolClin. Chem. 20, 470–5.
Bergman, W. (1958) Evolutionary concept of the sterols. InCholesterol, pp. 435–444. New York: Academic Press.
Brockerhoff, H. &Jensen, R. (1974)Lipolytic Enzymes, pp. 177–193. New York: Academic Press.
Elias, P., Goerke, J. &Friend, D. (1978) Freeze-fracture identification of sterol-digitonin complexes in cell and liposome membranes.J. Cell Biol. 78, 577–96.
Emeis, J., Van Gent, C. &Van Sabben, C. (1977) An enzymatic method for the histochemical localization of free and esterified cholesterol separately.Histochem. J. 9, 197–204.
Fruhling, J., Penasse, W., Sand, G., Morena, E. &Claude, A. (1970) Etude comparative par microscopie électronique des réactions cytochimiques de la digitonine avec le cholesterol et d'autres lipides présents dans les cellules de la corticosurrénale.Archs. int. Physiol. 78, 997–8.
Higgins, J., Florendo, N. &Barrnett, R. (1973) Localization of cholesterol in membranes of erythrocyte ghosts.J. Ultrastruct. Res. 42, 66–81.
Hirai, K. (1971) Comparison between 3,3′-diaminobenzidine and auto oxidized 3,3′-diaminobenzidine in the cytochemical demonstration of oxidative enzymes.J. Histochem. Cytochem. 19, 434–42.
Kruth, H. S. &Vaughan, M. (1980) Quantification of low density lipoprotein binding and cholesterol accumulation by single human fibroblasts using fluorescence microscopy.J. Lipid Res. 21, 123–30.
Makita, T., Fujimoto, K. &Kiwaki, S. (1978) Application of clinical test kits to histochemistry. I. Enzymatic determination and staining of total cholesterol.Acta Histochem. Cytochem. 11, 142–50.
Moses, H., Davis, W., Rosenthal, A. &Garren, L. (1969) Adrenal cholesterol: Localization by electron-microscope autoradiography.Science 163, 1203–5.
Napolitano, L. M., Saland, L., Lopez, J., Sterzing, P. R. &Kelley, R. O. (1972) Localization of cholesterol in peripheral nerve: Use of H3 digitonin for electron microscopic autoradiography.Anat. Rec. 174, 157–64.
Ökrös, I. (1968) Digitonin reaction in electron microscopy.Histochemie 13, 91–6.
Robinson, J. M., &Karnovsky, M. J. (1980) Evaluation of the polyene antibiotic filipin as a cytochemical probe for membrane cholesterol.J. Histochem. Cytochem. 28, 161–8.
Scallen, T. &Dietert, S. (1969) The quantitative retention of cholesterol in mouse liver prepared for electron microscopy by fixation in a digitonin-containing aldehyde solution.J. Cell Biol. 40, 802–13.
Seligman, A., Shannon, W., Hoshino, Y. &Plapinger, R. (1973) Some important principles in 3,3′-diaminobenzidine ultrastructural cytochemistry.J. Histochem. Cytochem. 21, 756–8.
Szabo, D., Dzsinich, C. S. &Ökrös, I. (1971) Ultrastructural localization of adrenal cholesterol by autoradiography and digitonin reaction after cycloheximide-induced inhibition of corticosterone synthesis.Histochemie 27, 43–9.
Vermeer, B., Van Gent, C., De Bruijn, W. &Boonders, T. (1978) The effect of digitonin-containing fixatives on the retention of free cholesterol and cholesterol esters.Histochem. J. 10, 287–98.
Williamson, J. (1969) Ultrastructural localization and distribution of free cholesterol (3β-hydroxysterols) in tissues.J. Ultrastruct. Res. 27, 118–33.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Martin Jones, H., Miyai, K. Ultrastructural localization of cholesterol by enzyme histochemistry. Histochem J 13, 1017–1028 (1981). https://doi.org/10.1007/BF01002641
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF01002641