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NEW HISTOCHEMICAL TECHNIQUES FOR THE DEMONSTRATION OF TISSUE OXIDASE (CYTOCHROME OXIDASE)
1 National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland
A group of complex naphthols, as well as open chain and cyclic methylene compounds, were found to be useful new reagents for the demonstration of tissue oxidase in conjunction with N-phenyl-p-phenylenediamine. The methylene compounds constitute a new class of histochemical reagents.
N-alkyl amines (e.g. N,N-dimethyl arylamines) are toxic to the enzyme and are to be avoided. Complex amines are inhibitory and are likewise not recommended.
As with the original "Nadi" technique, the reaction was inhibited by cyanide, azide, and sulfide. The distribution of staining in tissues was essentially identical with all reagents. Although the reaction was augmented by cytochrome c, this expensive reagent is not neeeded with the present techniques.
The indoaniline and azamethine dyes produced are highly insoluble and result in permanent or relatively permanent preparations. Most of the dyes are capable of complexing with mercury, molybdenum, cobalt, nickel, cadmium, lead, uranium, and iron, and thus may have potential applications in electron microscopy.
Among the naphthols 1-hydroxy-2-naphthoic acid (IV), 1-hydroxy-2-acetonaphthone (V), and a derivative of 1-hydroxy-2-naphthanilide (IX) are very useful couplers. Useful methylene compounds are exemplified by 1-phenyl-3(m-nitrobenzamidopyrazolone) (XII), naphthol AS-LG (XVIII), and naphthol AS-L3G (XX). Use of methylene compounds results in highly stable incubating solutions. All substrates are commercially available and inexpensive.
Unfixed frozen sections mounded on glass slides were used. Considerable caution must be exercised in attempts to localize oxidative enzyme activity at the cytologiocal level in unfixed frozen sections. This is due to the disruption of mitochondria and other cytological constituents which may occur during freezing and thawing. An alternative technique was evolved. This consisted of incubating fresh tissue slices, freezedrying them, followed by embedding in paraffin. Sections were then cut, dewaxed, and mounted.
Preliminary results indicate that tissue oxidase may also be demonstrated using a mixture of amines (e.g., N-phenyl-p-phenylenediamine and 2-methoxy-N-phenylenediamine) which are capable of forming complex azine dyes. The possibility of demonstrating other enzymes of the oxidase dehydrogenase complex warrants further investigation.
Submitted on August 28, 1958
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