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. 1961 Feb 1;9(2):415–427. doi: 10.1083/jcb.9.2.415

FORMALIN FIXATION IN THE CYTOCHEMICAL DEMONSTRATION OF SUCCINIC DEHYDROGENASE OF MITOCHONDRIA

Donald G Walker 1, Arnold M Seligman 1
PMCID: PMC2225007  PMID: 13782700

Abstract

A variety of established methods for protecting mitochondria were tested on rat duodenal epithelium during the histochemical assay for succinic dehydrogenase. The use of sucrose at isotonic or hypertonic concentrations, 7.5 per cent polyvinylpyrrolidone, divalent cations, physiological salt solutions, phenazine methosulfate, coenzyme Q10, and menadione failed to improve the quality of the histochemical preparation once fresh frozen sections were prepared. However, preservation of mitochondrial integrity with little diminution in succinic dehydrogenase activity was obtained by fixing tissue slices (less than 1 mm. in thickness) in 8 per cent unneutralized, aqueous formaldehyde from 8 to 16 minutes at from 5° to 10°C. prior to freezing. To offset the inhibition of enzymatic activity it was necessary to extend the incubation period by 10 to 15 minutes. Two-micron-thick sections were easily obtained from the frozen blocks of such fixed tissue and incubated in the unmodified Nitro—BT-succinate medium. Once the optimum conditions for fixation of intestinal epithelium were determined, many other tissues were subjected to the same procedure. From the morphological standpoint the appearance of the mitochondria in these histochemical preparations compares favorably with the results obtained using the classical Regaud iron-hematoxylin staining procedure. With most tissues, the results are superior to those with fresh frozen sections. However, results with muscle, sperm, and kidney tubular epithelium are not as strikingly improved as with gut and liver.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. BERGMAN R. A., WALKER D. G. The cytochemical localization of oxidative enzymatic activity and glycogen in frog striated muscle. Bull Johns Hopkins Hosp. 1959 Apr;104(4):179–198. [PubMed] [Google Scholar]
  2. GODDARD J. W., SELIGMAN A. M. Histochemical demonstration of succinic dehydrogenase in rat hepatoma. Cancer. 1953 Mar;6(2):385–389. doi: 10.1002/1097-0142(195303)6:2<385::aid-cncr2820060223>3.0.co;2-3. [DOI] [PubMed] [Google Scholar]
  3. GODDARD J. W., SELIGMAN A. M. Intracellular topography of succinic dehydrogenase in the thyroid of the albino rat. Anat Rec. 1952 Mar;112(3):543–559. doi: 10.1002/ar.1091120307. [DOI] [PubMed] [Google Scholar]
  4. HESS R., SCARPELLI D. G., PEARSE A. G. Cytochemical localization of pyridine nucleotide-linked dehydrogenases. Nature. 1958 May 31;181(4622):1531–1532. doi: 10.1038/1811531a0. [DOI] [PubMed] [Google Scholar]
  5. HESS R., SCARPELLI D. G., PEARSE A. G. The cytochemical localization of oxidative enzymes. II. Pyridine nucleotide-linked dehydrogenases. J Biophys Biochem Cytol. 1958 Nov 25;4(6):753–760. doi: 10.1083/jcb.4.6.753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. LEHNINGER A. L. Energy transformation in the cell. Sci Am. 1960 May;202:102–114. doi: 10.1038/scientificamerican0560-102. [DOI] [PubMed] [Google Scholar]
  7. NACHLAS M. M., MARGULIES S. I., SELIGMAN A. M. A colorimetric method for the estimation of succinic dehydrogenase activity. J Biol Chem. 1960 Feb;235:499–503. [PubMed] [Google Scholar]
  8. NACHLAS M. M., TSOU K. C., DE SOUZA E., CHENG C. S., SELIGMAN A. M. Cytochemical demonstration of succinic dehydrogenase by the use of a new p-nitrophenyl substituted ditetrazole. J Histochem Cytochem. 1957 Jul;5(4):420–436. doi: 10.1177/5.4.420. [DOI] [PubMed] [Google Scholar]
  9. NOVIKOFF A. B., MASEK B. Survival of lactic dehydrogenase and DPNH-diaphorase activities after formol-calcium fixation. J Histochem Cytochem. 1958 May;6(3):217–217. doi: 10.1177/6.3.217. [DOI] [PubMed] [Google Scholar]
  10. PEARSE A. G., SCARPELLI D. G., HESS R. A cytochemical study of the dehydrogenases of mitochondria and mitochondrial particulates by a monotetrazolium-cobalt chelation method. J Biophys Biochem Cytol. 1960 Apr;7:243–249. doi: 10.1083/jcb.7.2.243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. SCARPELLI D. G., PEARSE A. G. Physical and chemical protection of cell constituents and the precise localization of enzymes. J Histochem Cytochem. 1958 Sep;6(5):369–376. doi: 10.1177/6.5.369. [DOI] [PubMed] [Google Scholar]
  12. SIEKEVITZ P., PALADE G. E. A cytochemical study on the pancreas of the guinea pig. I. Isolation and enzymatic activities of cell fractions. J Biophys Biochem Cytol. 1958 Mar 25;4(2):203–218. doi: 10.1083/jcb.4.2.203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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. 1960 Jul;8:296–303. doi: 10.1177/8.4.296. [DOI] [PubMed] [Google Scholar]
  14. WITTER R. F., WATSON M. L., COTTONE M. A. Morphology and ATP-ase of isolated mitochondria. J Biophys Biochem Cytol. 1955 Mar;1(2):127–138. doi: 10.1083/jcb.1.2.127. [DOI] [PMC free article] [PubMed] [Google Scholar]

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