Summary
Freeze-drying and fluorescence microscopy techniques were combined to create a sensitive method for the visualization of the teratogenic dye, Trypan blue, in both protein-bound and free forms. In the development and initial application of this method, visceral yolk sacs of several gestational ages as well as normal appearing, 12-day embryos obtained from dye-injected rats were utilized. Observations on paraffinized sections of the yolk sac placentae demonstrated that only the protein-bound form of the dye exists in the yolk sac cavity whereas both forms of the dye exist in supranuclear regions of cells of the visceral endoderm. Paraffin sections of the normal appearing, 12-day embryos displayed the protein-bound form of dye within lumina of mid- and hind-gut, and both forms of dye in the primitive mucosa of mid- and hind-gut. The advantages of the method are derived not only from the use of fluorescence microscopy but also from the avoidance of solvents that are employed in more routine microtechniques.
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Beck, F., Lloyd, J.B.: An investigation of the relationship between foetal death and foetal malformation. J. Anat. 97, 555–564 (1963)
Cornell, R., Padykula, H.A.: A cytological study of intestinal absorption in the suckling rat. Amer. J. Anat. 125, 291–316 (1969)
Davis, H.W., Gunberg, D.L.: Trypan blue in the rat embryo. Teratology 1, 125–134 (1968)
Gillman, J., Gilbert, C., Gillman, T., Spence, I.: A preliminary report on hydrocephalus, spina bifida and other congenital anomalies in the rat produced by trypan blue. S. Afr. J. Med. Sci. 13, 47–90 (1948)
Greenhouse, G., Hamburgh, M.: Analysis of trypan blue induced teratogenesis in Rana pipiens embryos. Teratology 1, 61–73 (1968)
Hamberger, A., Hamberger, B.: Uptake of catecholamines and penetration of trypan blue after blood-brain barrier lesions Z. Zellforsch. 70, 386–392 (1966)
Hamburgh, M.: The embryology of Trypan blue induced abnormalities in mice. Anat. Rec. 119, 409–427 (1954)
Hamburgh, M., Erlich, M., Nathanson, G., Pesetsky, I.: Some additional observations relating to the mechanism of trypan blue induced teratogenesis. J. Exp. Zool. 192, 1–12 (1975)
Jollie, W.P.: Changes in the fine structure of the parietal yolk sac of the rat placenta with increasing gestational age. Amer. J. Anat. 122, 513–532 (1968)
Kormano, M.: Penetration of intravenous trypan blue into the rat testis and epididymis. Acta Histochem. (Jena) 30, 133–136 (1968)
Lloyd, J.B., Beck, F.: Evidence for a mechanism of teratogenesis involving inhibition of embryotrophic nutrition. Lab. Anim. 2, 171–180 (1968)
Lloyd, J.B., Beck, F.: Teratogenesis. In: Lysosomes in Biology and Pathology (Dingle, J.T., Fell, H.B., eds.), vol. 1, ch. 16, 433–449. Amsterdam: North-Holland Publishing Co. 1969
Lloyd, J.B., Beck, F., Griffiths, A., Parry, L.M.: The mechanism of action of acid bisazo dyes. In: The Interaction of Drugs and Subcellular Components in Animal Cells (Campbell, P.N., ed.), 171–200. London: Churchill 1968
Mulherkar, L.: The effects of trypan blue on chick embryos cultured in vitro. J. Embryol. Exp. Morph. 8, 1–5 (1960)
Nairn, R.C.: Direct protein tracing in experimental physiology and pathology. In: Fluorescent Protein Tracing (Nairn, R.C., ed.), 3rd ed., ch. 5, 95–110. Baltimore: The Williams and Wilkins Co. 1969
Padykula, H.A., Richardson, D.: A correlated histochemical and biochemical study of glycogen storage in the rat placenta. Amer. J. Anat. 112, 215–242 (1963)
Pearse, A.G.E.: Histochemistry: Theoretical and applied, 3rd ed., vol. 1, ch. 3, 27–58. Boston: Little, Brown and Co. 1968
Pearse, A.G.E.: Histochemistry. Theoretical and applied, 3rd ed., vol. 2., Edinburgh: Churchill Livingstone 1972
Pertussa, J.: A simple technique for the preservation of vital dyes in fixed and sectioned embryos. J. Embryol. Exp. Morph. 15, 131–132 (1966)
Rawson, R.A.: The binding of T-1824 and structurally related diazo dyes by the plasma proteins. Amer. J. Physiol. 138, 708–717 (1943)
Steinwall, O., Klatzo, I.: Selective vulnerability of the blood-brain barrier in chemically induced lesions. J. Neuropath. Exp. Neurol. 25, 542–559 (1966)
Telfer, W.H., Anderson, L.M.: Functional transformations accompanying the initiation of a terminal growth phase in the Cecropia moth oocyte. Dev. Biol. 17, 512–535 (1968)
Thilander, H.: Histologic technique for tissues stained with trypan blue. Anat. Anz. 115, 89–107 (1964)
Turbow, M.M.: Trypan blue induced teratogenesis of rat embryos cultivated in vitro. J. Embryol. Exp. Morph. 15, 387–395 (1966)
Udenfriend, S.: Fluorescence assay in biology and medicine, 223. New York: Academic Press 1962
Waddington, C.H., Carter, T.C.: A note on abnormalities induced in mouse embryos by Trypan blue. J. Embryol. Exp. Morph. 1, 167–180 (1953)
Waddington, C.H., Perry, M.M.: Teratogenic effects of trypan blue on amphibian embryos. J. Embryol. Exp. Morph. 4, 110–119 (1956)
Williams, W.L.: Vital staining of damaged liver cells. I. Reactions to acid azo dyes following acute chemical injury. Anat. Rec. 101, 133–147 (1948)
Williams, W.L., Frantz, M.: Histological technics in the study of vitally stained normal and damaged cells. Anat. Rec. 100, 547–559 (1948)
Wilson, J.G.: Teratogenic activity of several azo dyes chemically related to Trypan blue. Anat. Rec. 123, 313–333 (1955)
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Supported in part by the Oregon Heart Association and by the Medical Research Foundation of Oregon, grant 7513
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Davis, H.W., Sauter, R.W. Fluorescence of trypan blue in frozen-dried embryos of the rat. Histochemistry 54, 177–189 (1977). https://doi.org/10.1007/BF00492240
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DOI: https://doi.org/10.1007/BF00492240