New histochemical and cytochemical methods for the demonstration of cytochrome c oxidase and of cytochrome c-cytochrome oxidase system are described, using neotetrazolium chloride in the presence of p phenylenediamine with or without additional cytochrome c. These enable the cytochemical visualization of the sites of enzyme activity at the intracellular level in fresh cell suspensions and in fresh tissue blocks under aerobic
conditions, and permit the histochemical visualization of the distribution of the enzyme activity in various tissues in frozen tissue sections. The colorimetric estimation of the enzyme activity is also possible in the combination of the methods previously described.</p

Oda, Takuzo

Okazaki, Hiroaki

Seki, Shuji

Okayama University Scientific Achievement Repository

Acta Medica OkayamaVolume 12, Issue 4 1958 Article 3DECEMBER 1958New methods for the histochemical andcytochemical demonstrtion of cytochrome coxidase and of cytochrome c- cytochromeoxidase systemTakuzo Oda∗ Hiroaki Okazaki†Shuji Seki‡∗Okayama University,†Okayama University,‡Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.New methods for the histochemical andcytochemical demonstrtion of cytochrome coxidase and of cytochrome c- cytochromeoxidase system∗Takuzo Oda, Hiroaki Okazaki, and Shuji SekiAbstractNew histochemical and cytochemical methods for the demonstration of cytochrome c oxidaseand of cytochrome c-cytochrome oxidase system are described, using neotetrazolium chloride inthe presence of p phenylenediamine with or without additional cytochrome c. These enable thecytochemical visualization of the sites of enzyme activity at the intracellular level in fresh cellsuspensions and in fresh tissue blocks under aerobic conditions, and permit the histochemical vi-sualization of the distribution of the enzyme activity in various tissues in frozen tissue sections.The colorimetric estimation of the enzyme activity is also possible in the combination of the meth-ods previously described.∗Copyright c©OKAYAMA UNIVERSITY MEDICAL SCHOOLActa Med. Okayama 12, 302-309 (1958)NEW METHODS FOR THE HISTOCHEMICAL AND CYTOCHEMICALDEMONSTRTION OF CYTOCHROME C OXIDASE AND OFCYTOCHROME C - CYTOCHROME OXIDASE SYSTEMTakuzo OnA, Hiroaki OKAZAKI, and Shuji SEKIDepartment of Pathology, Okayama University Medical SchoolOkayama, Japan (Director: Prof. S. Seno)Received for publication, Oct. 9, 1958.For the histochemical detection of cytochrome oxidase, the Nadi reac-tion1 is frequently employed. This reaction consists in the formation ofindophenol blue from dimethyl-p-phenylenediamine and a-naphthol, andthe enzyme catalyzing the reaction has been called indophenol oxidase2 inthe field of biochemistry, which was identified with cytochrome oxidaseby KEILIN and HARTREE (1938)3. In the field of histochemistry, however,two kinds of enzymes had been known in the enzymes which could be de-tected using the Nadi reaction. The one is M-Nadi oxidase (GRAFF, ] 922)4,syn. stable oxidase (VON GIERKE, 1911)5, which is detected in formalinfixed myeloid white blood cells6 and a few kinds of tissue cells 7, and itis now considered as verdo-peroxidase8,9. The other is G-Nadi oxidase(GRAFF, 1922)4, syn. labile oxidase (VON GIERKE, 1911)5, which is de-tected in most of fresh tissue cells. LISON (1936)10 reported that the G-Nadioxidase must be identical with indophenol oxidasell • BRACHET (1944)12stated also that the Nadi reaction would take place by the action of cyto-chrome oxidase but at the same time the reaction product would be re-duced to its leucoderivative by the action of dehydrogenase, accordinglyno accurate picture on the enzyme distribution could be obtained by thisreaction. Moreover, as is well-known this reaction takes place easilyby autoxidation13 and by lipid peroxidesll ,14,15 even in the absence ofenzyme, and the reaction product secondarily stains lipids13.14 and othervarious elements in the cells!1. For these reasons, there are many objec-tions to the use of the reaction as a test for the histochemical detectionof cytochrome oxidase13 ,16.In our previous communications17,18, we described the new methodsfor colorimetric estimation of cytochrome c oxidase and of cytochrome c-cytochrome oxidase system in tissue homogenates, using neotetrazoliumchloride in the presence of p- phenylenediamine with or without ·additionalcytochrome c. It is the purpose of this study to establish a method for3021Oda et al.: New methods for the histochemical and cytochemical demonstrtionProduced by The Berkeley Electronic Press, 1958Histochemistry of Cytochrome Oxidase . 303the histochemical demonstration of the enzyme or enzyme system basingon the theory in the previous communications17,18,19.20.MATERrA L S AND METHODSFresh liver, kidney, heart and bone marrow tissues and cells fromdecapitated mice were used as materials. Incubation was carried out onfresh tissue blocks of about 1 to 2 mm. thick, and on frozen tissue sectionsof about 10 p. thick. Incubating medium for cytochrome c oxidase wasprepared immediately before incubation by combining equal volumes(mainly 0.2 ml. in each) of phosphate buffer (O.lM, pH 7.6) or sucrose solu-tion (1 M), p-phenylenediamine solution (p-PDA) (0.2M), neotetrazoliumchloride solution (0.2 %) and cytochrome c (10-~M). For cytochrome c-cytochrome oxidase system cytochrome c was omitted and equal volume ofphosphate buffer was added. For substrate contols p·PDA or both p-PDAand cytochrome c were omitted and equal volume of phosphate buffer wasadded. Endogenous dehydrogenase reaction conjugating at the stepsbelow antimycin A sensitive step20 could be almost completely eliminatedby freezing the tissue under -20 0 during 5 to 10 minutes before incuba-tion. As described in our previous communications18 ,20,2\ it is requiredto use the adequate amount of tissue comparing to the volume and con·centration of substrate solution for the quantitative reaction by thesemethods. Hence, about 40 mg. of tissue sections or a tissue block weredirectly put into the incubation medium of 0.8 ml.Incubation at 37°C was conducted aerobically or anaerobically for 30minutes to 1 hours. Then, cell suspensions were smeared and some of thetissue block were stamped on slides. Some of these specimens and tissuesections and blocks were washed with 0.33 M sucrose solution or physio-logic saline solution. The sections were mounted on slides. The smearedor stamped specimens were fixed with vapored formalin, and the sec-tioned specimens and the blocks were fixed with 10per cent formalin solu-tion. The blocks were sectioned by freezing or by carbowax embedding andthen mounted on slides. Most of these specimens were examined withoutcounter staining in glycerine immersion. The specimens was conservablefor a long time at least during one year. Some of the specimens werecounterstained with hematoxylin and eosin weakly or safranin O. For re-moving absolutely P-PDA or its oxidized product, the specimens werewashed with 0.1 M sulfuric acid after fixation.2Acta Medica Okayama, Vol. 12 [1958], Iss. 4, Art. 3http://escholarship.lib.okayama-u.ac.jp/amo/vol12/iss4/3304 T. ODA, H.OKAZAKl and S. SEKlRESULTS AND DISCUSSIONThe reactions for the enzyme and enzyme system in cell suspen-sions or tissue blocks resulted in a rich violet diformazan which was de-posited on mitochondria-like organellas in the cytoplasm in the form offine granules and rods. The reactions were completely negative in thenucleus. The distribution of the enzyme reations in cells was almost thesame as that of the succinoxidase system reaction1\l,22,23, but the activitywas not always parallel between the cytochrome c-cytochrome oxidasesystem and succinoxidase system in various kinds of cells18 ,21,22. The reac-tion in frozen tissue sections appeared less intensely and less distinctlythan the reaction in cell suspensions or tissue blocks. In this case, too,the reaction was completely negative in the nucleus.Distribution of enzyme activity in various kinds of tissues or cellswill be brieflY described in the explanation of figures.Specificity of the reaction in tissue homogenates was already provedin the previous communications18,2o but it was also tested in the presenthistochemical reaction: By the addition of potassium cyanide (IO-aM) buf-ferized at pH 7.6 with monobasic sodium phosphate in the incubationmedium, the histochemical reaction of the enzyme and enzyme systemseemed to be completely inhibited when weak endogenous reaction in liv-ing cells was eliminated. The specimens exposed to 80°C for 5 minutesbefore incubation were totally inactivated. Sodium malonate and anti-mycin A caused no inhibition. Anaerobic conditions enhanced the enzymereaction. P-PDA could be replaced by ascorbic acid, but the reactionusing p-PDA was more intense than the reaction using ascorbic acid. En-dogenous dehydrogenase reaction hardly appeared in frozen section butappeared weakly in fresh cell suspensions and tissue blocks. The endoge-nous reaction takes place conjugating at the step below the antimycin Asensitive step in the terminal electron transport system and at the otherreduction systems as presented in the previous communication20, and mostof the reaction could be eliminated by freezing the tissue before incuba-tion. As cytochrome c hardly p~netrates into living cells, it was difficultto demonstrate selectively cytochrome c oxidase activity irrespective ofthe amount of endogenous cytochrome c by the histochemical reaction infresh tissue blocks and cell suspensions, and therefore the reaction ofNT reduction by living cells in the presence of p-PDA with or withoutadditional cytochrome c are all considered to be responsible for cytochromec-cytochrome oxidase system activity in the tissues. In this case, it isconsidered that p-PDA reduces endogenous cytochrome c and the reduced3Oda et al.: New methods for the histochemical and cytochemical demonstrtionProduced by The Berkeley Electronic Press, 1958Histochemistry of Cytochrome Oxidase 305cytochrome c subsequently reduces NT by the action of cytochrome coxidase; in other words, this reaction takes place through co-operation ofcytochrome c and cytochrome c oxidase in the tissue 3. In frozen sectionsthe reaction was enhanced by the addition of cytochrome c, therefore inthis case the reaction with the addition of cytochrome c is considered tobe responsible for cytochrome c oxidase activity, as described in the pre-vious communication18• However, as the reaction due to the endogenouscytochrome c could hardly be eliminated even in frozen section, it will benecessary to compare with the control specimens without additional cyto-chrome c in order to examine the cytochrome c oxidase activity selective-ly, without any regard to the influence of the endogenous cytochrome c.SUMMARYNew histochemical and cytochemical methods for the demonstrationof cytochrome c oxidase and of cytochrome c-cytochrome oxidase systemare described, using neotetrazolium chloride in the presence of p-pheny-lenediamine with or without additional cytochrome c. These enable thecytochemical visualization of the sites of enzyme activity at the intracel-lular level in fresh cell suspensions and in fresh tissue blocks under aero-bic conditions, and permit the histochemical visualization of the distribu-tion of the enzyme activity in various tissues in frozen tissue sections.The colorimetric estimation of the enzyme activity is also possible in thecombination of the methods previously described.LITERATURE1. EHRLICH, P. : Das SauerstoffbedUrfnis der Organismen. Hirschwald, Berlin, 18852. KEILIN, D. : The action of sodium azide on cellular respiration and on some ca-talytic oxidation reactions. Proc. Roy. Soc. BioI. 121, 165-172, 19363. KEILIN, D. and HARTREE, E. F. : Cytochrome oxidase. Proc. Roy. Soc. Bioi. 125,171-186, 19364. GRAFF, S., Die mikromorphologischen Methoden der Fermentforschung imtierischen und pflanzlichen Organismus. Abderhalden Handb. Abt. IV, Teil 1,Heft 1, Methoden der Fermentforschung. 93-142, 19225. GIERKE, E. V. : Die oxidierende Zellfermente. Munch. med. Woschr. 58, 2315-2317, 19116. WINCKLER, F. : Der Nachweis von Oxydase in den Leukozyten mittels der Dime-thylparaphenylendiamin Alphanaphthol-Reaktion. Fol. Haem. 4, 323-328, 19077. SCHULTZE, W. H. : Die Oxydasereaktion an Gewebsschnitten und ihre BedeutungfUr die Pathologie. Ziegler, 45, 127-153, 19098. AGNER, K. Verdoperoxidase, a ferment isolated from leucocytes. Acta Physiol.Scand., Il, Suppl. 8, I, 19414Acta Medica Okayama, Vol. 12 [1958], Iss. 4, Art. 3http://escholarship.lib.okayama-u.ac.jp/amo/vol12/iss4/3306 T.OnA, H.OKAZAKI and S. SEKISkeletal muscle (m. soleus). Red muscle fibers are slender and stronglywhite muscle fibers are thick and weakly active. Between red andfibers, one or two kinds of muscle fibers of intermediate activity are9. SHIMIZU, M. : On the porphyrin enzymes in leucocytes. Acta Haem. fap. 21,(2), Suppl., 381-385, 195810. LISON. L. : Soc, Chim. bioI. 18, 185, 1936, cited from (11)11. LISON, L. : Histochimie et cytochimie animaIes, principles et midhodes. 2nd. ed.Guathier-Villars, Paris, 195312. BRAcHET, J. : Embryologie chimique. Masson, Paris, 194413. DIETRICH, A. : Naphtholblausynthese und Lipoidfarbung. Zbl. Path. 19, 3-6, 190814. GOMORI, G.: Microscopic Histochemistry. Univ. Chicago Press, 195215. PEARSE, A. G. E. : Histochemistry. Churchill, London, 195316. BRENNER, S. : South African f. M. Sc. 13, 320, 1947, cited from (11)17. OnA, T. : Cytochemical and biochemical studies on the terminal electron trans-port system. 1. Analytical studies on the colorimetric estimation of the acti-vities of the succinic dehydrogenase system and cytochrome oxidase. (in Japanesewith English abstract). Symp. Soc. Cell. Chem. 8, 157-172, 195818. OnA, T., SEKI, S., and OKAZKI, H. : New colorimetric methods for the estima-tion of cytochrome c oxidase and of cytochrome c-cytochrome oxidase system.Acta Med. Okayama 12, 293-301, 195819. OnA, T. : Cytochemical and biochemical studies on the terminal electron trans-port system n. Cytochemical demonstration of the succinic dehydrogenase systemand cytochrome oxidase in mitochondia with the electron microscope (in Japanesewith English abstract). Symp. Soc. Cell. Chem. 8, 173-190. 195820. OnA, T., and OKAZAKI, H. : An anlytical study of the reduction of neotetrazo-lium chloride by the terminal electron transport system. Acta Med. Okayama.12, 193-204, 195821. OnA, T., SEKI, S., SHIBATA, T., SAKAI, A., and OKAZAKI, H. :' Analytical studieson the colorimetric estimation of the activity of succinic dehydrogenase systemwith neotetrazolium chloride (in Japanese with English abstract). OkayamaIgakkai Zasshi 70, (1), 123-135, 195822. OnA, T., MATSUOKA, K. and OKAZAKI, H. : Histochemical and cytochemical studieson the succinic dehydrogenase system with three ditetrazolium salts, NT, Nitro-NT, and Nitro·BT. Acta Med. Okayama 13, in press23. OnA, T., SAKAI, and OKAZAKI, H. : Cytochemical demonstration of the sites ofactivity of the terminal electron transport system with the electron microscope.Acta Med. Okayama 12, 205-215, 1958EXPLANATION OF FIGURESAll figures show the reaction of cytochrome c-cytochrome oxidase system in va-rious tissues of the mouse. In Figs. 1-6, the reaction was conducted for one hourin tissue blocks, and then the blocks were fixed with 10 per cent formalin solution,embedded in carbowax, washed briefly with 0.1 M sulfuric acid, immersed in glycerinwithout counterstaining.Fig. 1. Cardiac muscle. Strong reaction appears as pigment granules depositedon mitochondria·like organellae between muscle fibrils in the cardiac muscle. Nucleiare negative.Fig. 2.active, whilewhite muscle5Oda et al.: New methods for the histochemical and cytochemical demonstrtionProduced by The Berkeley Electronic Press, 1958Histochemistry of Cytochrome Oxidase 307demonstrated. Pigments are deposited on mitochondria in rows between musclefibrils. The intensity of the reaction in muscle fibers is dependent on the numberand the activity of mitochondria contained in them, which differ distinctly amongred and white muscle fibers.Fig. 3. Liver. Pigment depositon on the organellae of the hepatic and Kupffercells showing the oval or rod-like shapes which the mitochondria may take. Nucleiare completely negative.Fig. 4. Kidney. Convoluted tubular epithelial cells are strongly active. Glo-meruli are very weakly active. In medulla, though they are not presented in thispicture, the Henle's tubules are strongly active but the collecting tubules are weak-ly active.Fig. 5. Small brain. Right half of this picture is the molecular layer and lefthalf is the granular layer. Two Purkinje cells are presented between two layers. Pig-ment granules are deposited in the cytoplasm and fibers of the cells but the nucleiare inactive. Unfavorable crystal formations are seen scatteringly in the molecularlayer. In granular layer, two kinds of cells, which differ distinctly in the activity,are presented.Fig. 6. Spleen. Reticulum cells are moderately active, while lymphocytes areweakly active.Fig. 7. Bone marrow cells of a mouse. The reaction was conducted in verysmall pieces of and cell suspensions of bone marrow tissues. After the reaction stampedor smeared specimen was prepared, fixed with formalin vapor, washed briefly with0.1 N sulfuric acid, washed with water, and counterstained weakly with hematoxylin-eosin. All blood cells with a single exception of matured red cells are active, thoughthe activity varies according to the kind of cells and grade of maturation. Twogranulocytes, a reticulocyte and a red cell are presented. The reaction appears onmitochondria in the cytoplasm of whit!:: blood cells. Reticulocytes also contain mi-tochondria and the enzyme activity is demonstrated.Fig. 8. Ascitic hepatoma cells of a rat. The reaction was conducted in cellsuspensions, and then smeared specimen was prepared, immersed in glycerin withoutcounterstain. The reaction appears on mitochondria in the cytoplasm. The activityof cancer cells is very irregular and differs according to the origin and the gradeof maturation. The reactions even in one cell being also irregular, strongly andweakly active mitochondria are mingled.6Acta Medica Okayama, Vol. 12 [1958], Iss. 4, Art. 3http://escholarship.lib.okayama-u.ac.jp/amo/vol12/iss4/3308 T.ODA H, .OKAZAKI and S. SEKI7Oda et al.: New methods for the histochemical and cytochemical demonstrtionProduced by The Berkeley Electronic Press, 1958Histochemistry of Cytochrome Oxidase 309Fig. 5.Fig. 7.Fig. 6.Fig. 8.8Acta Medica Okayama, Vol. 12 [1958], Iss. 4, Art. 3http://escholarship.lib.okayama-u.ac.jp/amo/vol12/iss4/3

New methods for the histochemical and cytochemical demonstrtion of cytochrome c oxidase and of cytochrome c- cytochrome oxidase system

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