Summary
A technique for the cytochemical demonstration of peroxidase activity in unfixed guinea-pig thyroid tissue is described in this paper. The substrate 3,3′-diaminobenzidine tetrahydrochloride (DAB) is oxidized by the peroxidase to form an insoluble reaction product. Optimal results were obtained after 20 min incubation at 37° C in reaction medium containing 1.4mm DAB (in 0.1m Tris-HCl) and 0.15mm hydrogen peroxide at pH 8.0. Peroxidase activity was seen in the thyroid follicle cells as a diffuse brown reaction product (which was more dense and granular in erythrocytes). The enzyme activity was quantified using a scanning-integrating microdensitometer, and the effects of two specific peroxidase inhibitors were evaluated. Both 3-amino-1,2,4-triazole and methimazole inhibited peroxidase activity in the follicle cells (enzyme activity was still seen in the erythrocytes), maximal inhibition occurring at 10mm. Stimulation of peroxidase in the thyroid was observedin vivo (1 I.U. TSH administered every 8 h for two days), with the maximal stimulation occurring after 1 day.
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Anderson, W. A., Trantalis, J. &Kang, Y. h. (1975) Ultrastructural localisation of endogenous mammary gland peroxidase during lactogenesis in the rat; results after tannic acid-formaldehyde-glutaraldehyde fixation.J. Histochem. Cytochem. 23, 295–302.
Bitensky, L., Alaghband-Zadeh, J. &Chayen, J. (1974) Studies on thyroid stimulating hormone and the long acting thyroid stimulator.Clin. Endocr. (Oxford) 3, 363–74.
Chayen, J., Bitensky, L. &Butcher, R. G. (1973)Practical Histochemistry. London: Wiley.
Chayen, J., Gilbert, D. M., Robertson, W. R., Bitensky, L. &Besser, G. M. C. (1980) A cytochemical section bioassay for thyrotropin.J. Immunoassay 1, 1–13.
Coval, M. L. &Taurog, A. (1967) Purification and iodinating activity of hog thyroid peroxidase.J. biol. Chem. 242, 5510–23.
De Groot, L. J. &Niepomniszcze, H. (1977) Biosynthesis of thyroid hormones: basic and clinical aspects.Metabolism 26, 665–718.
Ealey, P. A. &Smyth, P. P. A. (1980) Validation of the cytochemical section bioassay for thyroid stimulating antibodies.J. Immunoassay 1, 175–94.
Ealey, P. A., Marshall, N. J. &Ekins, R. P. (1981) Time-related thyroid stimulation by thyrotropin and thyroid-stimulating antibodies, as measured by the cytochemical section bioassay.J. clin Endocr. Metab. 52, 483–7.
Fahimi, H. D. (1979) An assessment of the DAB methods for the cytochemical detection of catalase and peroxidase.J. Histochem. Cytochem. 27, 1365–6.
Fahimi, H. D. &Herzog, V. (1973) A colorimetric method for measurement of the (peroxidase-mediated) oxidation of 3,3′-diaminobenzidine.J. Histochem. Cytochem. 21, 499–503.
Graham, R. C. &Karnovsky, M. J. (1966) The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique.J. Histochem. Cytochem. 14, 291–302.
Herzog, V. &Fahimi, H. D. (1976) Intracellular distinction between peroxidase and catalase in exocrine cells of rat lacrimal gland: a biochemical and cytochemical study.Histochemie 46, 273–86.
Herzog, V. &Miller, F. (1972) The localisation of endogenous peroxidase in the lacrimal gland of the rat during postnatal development: electron microscopy, cytochemical and histochemical studies.J. Cell Biol. 53, 662–80.
Hosoya, T., Matsukawa, S. &Kurata, Y. (1972) Cytochemical localisation of peroxidase in the follicular cells of pig thyroid gland.Endocr. jap. 19, 359–69.
Ljunggren, J. G. &Akeson, A. (1968) Solubilisation, isolation and identification of a peroxidase from the microsomal fraction of beef thyroid.Archs. Biochem. Biophys. 127, 346–53.
Nagataki, S., Uchimura, H., Masuyama, Y. &Nakao, K. (1973) Thyrotropin and thyroidal peroxidase activity.Endocrinology 92, 363–71.
Nakane, P. &Pierce, G. B. (1967) Enzyme-labelled antibodies for light and electron microscopic localisation of tissue antigens.J. Cell Biol. 33, 307–18.
Novikoff, A. B., Novikoff, P. M., Davis, C. &Quintana, N. (1972) Studies on microperoxisomes II. A cytochemical method for light and electron microscopy.J. Histochem. Cytochem. 20, 1006–23.
Novikoff, A. B., Novikoff, P. M., Ma, M., Shin, W.-Y. &Quintana, N. (1974) Cytochemical studies of secretory and other granules associated with the endoplasmic reticulum in rat thyroid epithelial cells.Adv. Cytopharmacol. 2, 349–68.
Robbins, D., Fahimi, H. d. &Cotran, R. s. (1971) Fine structural cytochemical localisation of peroxidase activity in rat peritoneal cells: Mononuclear cells, eosinophils and mast cells.J. Histochem. Cytochem. 19, 571–5.
Rosene, D. l. &Mesulam, M. (1978) Fixation variables in horseradish peroxidase neurohistochemistry 1. The effects of fixation time and perfusion procedures upon enzyme activity.J. Histochem. Cytochem. 26, 28–39.
Strum, J. m. &Karnovsky, M. j. (1970) Cytochemical localisation of endogenous peroxidase in thyroid follicular cells.J. Cell Biol. 44, 655–66.
Tice, L. w. &Wollman, S. h. (1972) Ultrastructural localisation of peroxidase activity on some membranes of the typical thyroid epithelial cell.Lab. Invest. 26, 63–73.
Yamamoto, K. &De Groot, L. J. (1974) Peroxidase and NADPH cytochrome C reductase activity during thyroid hyperplasia and involution.Endocrinology 95, 606–12.
Yip, C. (1966) Properties of a peroxidase purified from beef thyroid tissues.Biochim. biophys. Acta 128, 262–71.
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Ealey, P.A., Henderson, B. & Loveridge, N. A quantitative study of peroxidase activity in unfixed tissue sections of the guinea-pig thyroid gland. Histochem J 16, 111–122 (1984). https://doi.org/10.1007/BF01003543
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DOI: https://doi.org/10.1007/BF01003543