Summary
The present investigation was undertaken in order to establish an optimal tissue pretreatment and an optimal incubation medium for the histochemical demonstration of succinate dehydrogenase (E.C. 1.3.99.1). The investigations were performed on steroid producing (testicle, adrenal gland) and steroid dependent (Fallopian tube) tissues. We studied the influences of formalin fixation, acetone, magnesium ions, cyanides, electron carriers (phenazine methosulfate, menadione, coenzyme Q10), osmolarity, substrate concentration and inhibitors (oxalacetate, oxalate, malonate, 4-chloromercuribenzoic acid). The following procedure yields blameless morphological integrity and enzyme localization as well as optimal SDH-activity:
Freezing of tissue cubes (diameter less than 5 mm) in propane cooled with liquid nitrogen or in melting freon.
Incubation of 5 μm cryostat sections in narrow jars in the following medium (38.5 ml):
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- 10 ml of 0.2 M sodium phosphate buffer pH 7.6 (52 mM).
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- 18 mg tetranitro-BT in 0.5 ml dimethylformamide and aqua bidest. ad 10 ml (0.5 mM).
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- 2.6 mg KCN in 16 ml aqua bidest. (1 mM).
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- 540 mg succinate (disodium salt, hexahydrate) in 2 ml aqua bidest. (52 mM).
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- 3 mg PMS (phenazine methosulfate) in 0.5 ml aqua bidest. (0.25 mM).
The incubation medium has an osmolarity of 440 mosm.
The incubation is carried out for 10 min at 37° C in darkness.
To avoid non specific formazan deposits in lipid containing tissues a preincubation of the cryostat sections in 100% acetone at −22° C or −40° C for 7–10 min and an incubation time of 20–30 min is recommended.
Control incubations adduced proof at the specificity of the SDH demonstration. Parallel incubation without PMS in order to determine indirectly the content of endogenous CoQ10 is further recommended.
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References
Andersen, H., Hoyer, P.E.: Studies in succinate dehydrogenase histochemistry. Histochemie 35, 173–188 (1973)
Bergmeyer, H.U.: Methoden der enzymatischen Analyse. 2. Auflage Bd. I. Weinheim/Bergstraße: Verlag Chemie 1970
Bernath, P., Singer, T.P.: Succinic dehydrogenase. In: Methods in enzymology, ed. S.P. Colowick and N.O. Kaplan, Vol. 5, p. 597, New York: Academic Press 1962
Crane, F., Fechner, W., Ambe, K.: Two neutral lipids from mitochondria specifically required for coenzyme Q function. Arch. Biochem. 81, 277 (1959)
Deane, H.W., Barrnett, R.J., Seligman, A.M.: Enzymes. In: Handbuch der Histochemie, hrsg. von W. Graumann und K. Neumann, Vol. VII, part 1 a. Stuttgart: Gustav Fischer 1960
Egger, G.: On the methods for the electron microscopic demonstration of succinate-tetrazolium salt reductase with TC-NBT. Histochemie 33, 333–339 (1973)
Ernster, L., Kuylenstierna, B.: Outer membrane of mitochondria. In: Racker E. (ed.): Membranes of mitochondria and chloroplasts. ACS Monograph 165, p. 171–212. New York-Cincinnati-Toronto-London-Melbourne: Van Nostrand Reinhold 1970
Farber, E., Bueding, E.: Histochemical localization of specific oxidative enzymes. V. The dissociation of succinic dehydrogenase from carriers by lipase and the specific histochemical localization of the dehydrogenase with phenazine methosulfate and tetrazolium salts. J. Histochem. Cytochem. 4, 357–362 (1956)
Hardonk, M.J.: The use of phenazinemethosulphate as an electron carrier in the histochemical demonstration of dehydrogenases. Histochemie 4, 563–568 (1965)
Hashimoto, T., Kaluza, J.S., Burstone, M.S.: The effect of menadione and phenazine methosulfate on the tetrazolium reduction system under histochemical conditions. J. Histochem. Cytochem. 12, 797–804 (1964)
Hoch, F.L., Vallee, B.L.: Effects of adenosine triphosphate and metals on electron transport system in mitochondria. Nature 176, 256–257 (1955)
Holt, S.J.: Factors governing the validity of staining methods for enzymes and their bearing upon the Gomori acid phosphatase technique. Exp. Cell Res. Suppl. 7, 1–27 (1959)
Horwitz, C.S., Benitez, L., Bray, M.: The effect of coenzyme Q on the histochemical succinic tetrazolium reductase reaction: a histochemical study. J. Histochem. Cytochem. 15, 216–224 (1967)
Lehninger, A.L.: Biochemistry, the molecular basis of cell structure and function, 2nd ed. New York: Worth Publishers 1977
Lester, R., Fleischer, S.: The respiratory activity of acetone extracted beef-heart mitochondria. Role of coenzyme Q and other lipids. Biochem. Biophys. Acta 47, 358–377 (1961)
Lin, H.Y., Baker, B.L.: The influence of technique and hormones on histochemical demonstration of enzymes in intestinal epithelium. J. Histochem. Cytochem. 11, 349–364 (1963)
Lojda, Z., Gossrau, R., Schiebler, T.H.: Enzymhistochemische Methoden. Berlin-Heidelberg-New York: Springer-Verlag 1976
Massey, V., Singer, T.P.: Studies on succinic dehydrogenase. VI. The reactivity of beef heart succinic dehydrogenase with electron carriers. J. biol. Chem. 229, 755–762 (1957)
Novikoff, A., Shin, W., Drucker, J.: Cold acetone fixation for enzyme localization in frozen sections. J. Histochem. Cytochem. 8, 37–40 (1960)
Pearse, A.G.E.: Histochemistry, theoretical and applied, 3rd. ed., vol. 2. London: Churchill Livingstone 1972
Racker, E.: Mechanism of action and properties of pyridine nucleotide-linked enzymes. Physiol. Rev. 35, 1–56 (1955)
Singer, T.P., Kearney, E.B.: Solubilization, assay, and purification of succinic dehydrogenase. Biochem. Biophys. Acta 15, 151–153 (1954)
Tyler, D.B.: Effect of metal ions and adenosine triphosphate on an oxalacetate inhibited succinooxydase activity. J. biol. Chem. 216, 395–403 (1955)
Walker, D.G., Seligman, A.M.: Formalin fixation in the cytochemical demonstration of succinic dehydrogenase of mitochondria. J. biophys. biochem. Cytol. 9, 415–427 (1961)
Walker, D.G., Seligman, A.M.: The use of formalin fixation in the cytochemical demonstration of succinic and DPN- and TPN- dependent dehydrogenases in mitochondria. J. Cell Biol. 16, 455–469 (1963)
Wattenberg, L.W., Leong, J.L.: Effects of coenzyme Q10 and menadione on succinic dehydrogenase activity as measured by tetrazolium salt reduction. J. Histochem. Cytochem. 8, 296–303 (1960)
Wolman, M., Bubis, J.J.: Ubiquinone and phospholipids as limiting factors in the histochemical demonstration of succinic dehydrogenase activity. J. Histochem. Cytochem. 15, 79–82 (1967)
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Kugler, P., Wrobel, KH. Studies on the optimalisation and standardisation of the light microscopical succinate dehydrogenase histochemistry. Histochemistry 57, 47–60 (1978). https://doi.org/10.1007/BF00507355
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DOI: https://doi.org/10.1007/BF00507355