Synopsis
A quantitative histochemical method has been developed for the determination of the rate of formation of azo dye in an immediate coupling reaction for acid phosphatase. Predetermined areas of rat tail epidermis were continuously monitored by a photomultiplier. It is postulated that the rate of darkening is related to the rate of dye formation and that this in turn is dependent on the degree of enzyme activity in the area of tissue under observation. Regression coefficients were calculated for two different regions of rat tail epidermis. It was found that there was a significant difference between the transitional and lower epidermal zones:p<0.01 for 5 degrees of freedom.
An aqueous model system showed that there was a relationship between the rate of dye formation and enzyme concentration. At present it is not certain whether this method can be used for the absolute estimation of enzyme in tissues, but it does appear to be satisfactory as a comparative technique.
A slowing of the rate of reaction with time was clearly demonstrated and the greater the initial enzyme activity, the greater the rate of slowing. It is thought that this might be due to enzyme inhibition resulting from the azo dye formation.
It is suggested that this method is relatively unaffected by variations in section thickness and this is a great advantage when cryostat sections are used. However, compression of tissue during sectioning does produce inaccuracies.
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References
Baxter, R. &Danielli, J. F. (1955). A quantitative cytochemical method for estimating alkaline phosphatase activity.Proc. R. Soc. B144, 412–26.
Caspersson, T. (1932). Die quantitative Bestimmung von Thymonucliensäuremittels fuchsinschweslige Säuere.Biochem. Z. 253, 97–111.
Defendi, V. &Pearse, A. G. E. (1955). Significance of coupling rate in histochemical azo-dye methods for enzymes.J. Histochem. Cytochem. 3, 203–11.
Felgenhauer, K. &Glenner, G. G. (1966). Quantitation of tissue-bound renal amino peptidase by a microdensitometric technique.J. Histochem. Cytochem. 14, 53–63.
Friede, R. L. &Mechthilde, K. (1965). Quantitative tests on histochemical methods for phosphoesterases in brain.J. Histochem. Cytochem. 13, 125–32.
Glenner, G. G. (1965). Enzyme histochemistry. In:Neurohistochemistry (ed. Adams, C. W. M.), p. 109. Amsterdam; Elsevier.
Gunther, C. &Glick, D. (1967). Determination of histaminase activity in histological samples and its quantitative distribution in intact human placenta and uterus.J. Histochem. Cytochem. 15, 431–5.
Hiraoka, T. (1957). Feulgen nuclear reaction I. Quantitative extraction of fuchsin from Feulgen stained nucleoprotein.J. biophys. biochem. Cytol. 3, 625–9.
Holt, S. J. &O'Sullivan, D. G. (1958). Studies in enzyme cytochemistry.Proc. R. Soc. B148, 465–80.
Hopsu, V. K. &McMillan, J. P. (1964). Quantitative characterisation of histochemical enzyme system.J. Histochem. Cytochem. 12, 315–24.
Jarrett, A. (1963). Some contributions of fluorescence microscopy to the histochemistry of the skin.Annls Histochim. 8, supp. 1, 47–50.
Jarrett, A. (1968). Acid nucleases in human skin.J. invest. Derm. 49, 443–8.
Jarrett, A. & Bligh, A. (1960). A photometric method for the determination of nuclear proteins at histological levels.Proc. Ist. Int. Cong. Med. Photography, Dusseldorf.
Jarrett, A. &Spearman, R. I. C. (1964).Histochemistry of the Skin: Psoriasis. London: English Universities Press.
Jarrett, A. &Spearman, R. I. C. (1967). Keratinization.Derm. Digest 6, 43–53.
Jarrett, A., Spearman, R. I. C. &Hardy, J. A. (1959). Histochemistry of keratinization.Br. J. Derm. 71, 277–96.
McCabe, M., Maple, A. J. &Jones, G. R. N. (1965). Variations in dehydrogenase and neotetrasolium reductase (diaphorase) activities in various buffers studied quantitatively in frozen sections.J. Histochem. Cytochem. 13, 541–6.
Myrsky, A. E. &Ris, H. (1951). The desoxyribonucleic acid content of animals. Evolutionary significance.J. gen. Physiol. 34, 451–62.
Muir, A. V., Calman, K. C., Thomson, J., Macsween, M. P., Milne, J. A., Chakrahorty, J. &Grant, J. K. (1968). Hydroxysteroid dehydrogenase activity in human skin II. Factors influencing hydroxysteroid dehydrogenase activity in sebaceous glands of human skin.Br. J. Derm. 80, 652–6.
Nordstrom, C., Dahlqvist, A. &Lars, J. (1967). Quantitative determination of enzymes in different parts of the villi and crypts of rat small intestine.J. Histochem. Cytochem. 15, 713–21.
Pearse, A. G. E. (1954). Azo-dye methods in enzyme histochemistry.Intern. Rev. Cytology 3, 329–58.
Pearse, A. G. E. (1968).Histochemistry, 3rd Ed., Vol. 1, p. 491. London: Churchill.
Persijn, J. P. &Van Duijn, P. (1961). Studies of the Feulgen Reaction with the Aid of DNA Incorporated-cellulose Films.Histochemie 2, 283–97.
Swift, H. & Rasch, E. (1956). InPhysical techniques in biological research. (eds. Oster, G. and Pollister, A. W.), p. 553.
Taljedal, L., Hellman, B. &Hallerstrom, C. (1964). Quantitative studies on isolated pancreatic islets of mammals,J. Histochem. Cytochem. 12, 491–7.
Thaler, M. M. &Kissane, J. M. (1966). Quantitative distribution of lactic dehydrogenase in the regenerating liver lobule of the rat.J. Histochem. Cytochem. 14, 495–500.
Vallyathan, N. V., Cherian, K. M. &George, J. C. (1964). Histochemical and quantitative changes in glycogen and phosphorylase during disuse atrophy of the pigeon pectoralis.J. Histochem. Cytochem. 12, 721–8.
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Jarrett, A., Please, N.W. A quantitative histochemical technique for the estimation of azo dye coupling reactions. Histochem J 2, 297–313 (1970). https://doi.org/10.1007/BF01004998
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DOI: https://doi.org/10.1007/BF01004998