Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1977 Sep 1;74(3):992–1015. doi: 10.1083/jcb.74.3.992

Localization of thyroglobulin antigenicity in rat thyroid sections using antibodies labled with peroxidase or (125)I-radioiodine

J Paiement, CP Leblond
PMCID: PMC2110099  PMID: 71304

Abstract

In the hope of localizing thyroglobulin within focullar cells of the thyroid gland, antibodies raised against rat thyroglobulin were labeled with the enzyme horseradish peroxidase or with (125)I-radioiodine. Sections of rat thyroids fixed in glutaraldehyde and embedded in glycol methacrylate or Araldite were placed in contact with the labeled antibodies. The sites of antibody binding were detected by diaminobenzidine staining in the case of peroxidase labeling, and radioautography in the case of 125(I) labeling. Peroxidase labeling revealed that the antibodies were bound by the luminal colloid of the thyroid follicles and, within focullar cells, by colloid droplets, condensing vacuoles, and apical vesicles. (125)I labeling confirmed these findings, and revealed some binding of antibodies within Golgi saccules and rough endoplasmic reticulum. This method provides a visually less distinct distribution than peroxidase labeling, but it allowed ready quantitation of the reactions by counts of silver grains in the radioautographs. The counts revealed that the concentration of label was similar in the luminal colloid of different follicles, but that it varied within the compartments of follicular cells. A moderate concentration was detected in rough endoplasmic reticulum and Golgi saccules, whereas a high concentration was found in condensing vacuoles, apical vesicles, and in the luminal colloid. Varying amounts of label were observed over the different types of colloid droplets, and this was attributed to various degrees of lysosomal degradation of thyroglobulin. It is concluded that the concentration of thyroglobulin antigenicity increases during transport from the ribosomal site of synthesis to the follicular colloid, and then decreases during the digestion of colloid droplets which leads to the release of the thyoid hormone.

Full Text

The Full Text of this article is available as a PDF (6.3 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Avrameas S., Ternynck T. Peroxidase labelled antibody and Fab conjugates with enhanced intracellular penetration. Immunochemistry. 1971 Dec;8(12):1175–1179. doi: 10.1016/0019-2791(71)90395-8. [DOI] [PubMed] [Google Scholar]
  2. BERSON S. A., YALOW R. S. Radiochemical and radiobiological alterations of I 131-labeled proteins in solution. Ann N Y Acad Sci. 1957 Aug 30;70(1):56–68. doi: 10.1111/j.1749-6632.1957.tb35377.x. [DOI] [PubMed] [Google Scholar]
  3. Bauer W. C., Meyer J. S. Origin and fate of colloid droplets of the thyroid gland. A study using iodine-125 electron microscopy autoradiography. Lab Invest. 1965 Oct;14(10):1795–1808. [PubMed] [Google Scholar]
  4. Bilstad J. M., Edelhoch H., Lippoldt R., Rall J. E., Salvatore G. Isolation and characterization of discrete fragments of 27 S thyroid iodoprotein. Arch Biochem Biophys. 1972 Jul;151(1):341–350. doi: 10.1016/0003-9861(72)90505-x. [DOI] [PubMed] [Google Scholar]
  5. Björkman U., Ekholm R., Elmqvist L. G., Ericson L. E., Melander A., Smeds S. Induced unidirectional transport of protein into the thyroid follicular lumen. Endocrinology. 1974 Dec;95(6):1506–1517. doi: 10.1210/endo-95-6-1506. [DOI] [PubMed] [Google Scholar]
  6. Boorsma D. M., Kalsbeek G. L. A comparative study of horseradish peroxidase conjugates prepared with a one-step and a two-step method. J Histochem Cytochem. 1975 Mar;23(3):200–207. doi: 10.1177/23.3.47869. [DOI] [PubMed] [Google Scholar]
  7. COONS A. H., KAPLAN M. H. Localization of antigen in tissue cells; improvements in a method for the detection of antigen by means of fluorescent antibody. J Exp Med. 1950 Jan 1;91(1):1–13. doi: 10.1084/jem.91.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Clyne D. H., Norris S. H., Modesto R. R., Pesce A. J., Pollak V. E. Antibody enzyme conjugates: the preparation of intermolecular conjugates of horseradish peroxidase and antibody and their use in immunohistology of renal cortex. J Histochem Cytochem. 1973 Mar;21(3):233–240. doi: 10.1177/21.3.233. [DOI] [PubMed] [Google Scholar]
  9. Cuatrecasas P. Protein purification by affinity chromatography. Derivatizations of agarose and polyacrylamide beads. J Biol Chem. 1970 Jun;245(12):3059–3065. [PubMed] [Google Scholar]
  10. Ekholm R., Engström G., Ericson L. E., Melander A. Exocytosis of protein into the thyroid follicle lumen: an early effect of TSH. Endocrinology. 1975 Aug;97(2):337–346. doi: 10.1210/endo-97-2-337. [DOI] [PubMed] [Google Scholar]
  11. FELDMAN J. D., VAZQUEZ J. J., KURTZ S. M. Maturation of the rat fetal thyroid. J Biophys Biochem Cytol. 1961 Nov;11:365–383. doi: 10.1083/jcb.11.2.365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Farquhar M. G., Palade G. E. Cell junctions in amphibian skin. J Cell Biol. 1965 Jul;26(1):263–291. doi: 10.1083/jcb.26.1.263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fertuck H. C., Salpeter M. M. Quantitation of junctional and extrajunctional acetylcholine receptors by electron microscope autoradiography after 125I-alpha-bungarotoxin binding at mouse neuromuscular junctions. J Cell Biol. 1976 Apr;69(1):144–158. doi: 10.1083/jcb.69.1.144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Frati L., Bilstad J., Edelhoch H., Rall J. E., Salvatore G. Biosynthesis of the 27 S thyroid iodoprotein. Arch Biochem Biophys. 1974 May;162(1):126–134. doi: 10.1016/0003-9861(74)90111-8. [DOI] [PubMed] [Google Scholar]
  15. GLAUERT A. M., GLAUERT R. H. Araldite as an embedding medium for electron microscopy. J Biophys Biochem Cytol. 1958 Mar 25;4(2):191–194. doi: 10.1083/jcb.4.2.191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. GREENWOOD F. C., HUNTER W. M., GLOVER J. S. THE PREPARATION OF I-131-LABELLED HUMAN GROWTH HORMONE OF HIGH SPECIFIC RADIOACTIVITY. Biochem J. 1963 Oct;89:114–123. doi: 10.1042/bj0890114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Graham R. C., Jr, Karnovsky M. J. 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. 1966 Apr;14(4):291–302. doi: 10.1177/14.4.291. [DOI] [PubMed] [Google Scholar]
  18. Haddad A., Smith M. D., Herscovics A., Nadler N. J., Leblond C. P. Radioautographic study of in vivo and in vitro incorporation of fucose-3H into thyroglobulin by rat thyroid follicular cells. J Cell Biol. 1971 Jun;49(3):856–877. doi: 10.1083/jcb.49.3.856. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Haeberli A., Bilstad J., Edelhoch H., Rall J. E. Elementary chain composition of guinea pig thyroglobulin. J Biol Chem. 1975 Sep 25;250(18):7294–7299. [PubMed] [Google Scholar]
  20. Hayden L. J., Shagrin J. M., Young J. A. Micropuncture investigation of the anion content of colloid from single rat thyroid follicles. A micromethod for the simultaneous determination of iodide and chloride in nanomole quantities. Pflugers Arch. 1970;321(2):173–186. doi: 10.1007/BF00586371. [DOI] [PubMed] [Google Scholar]
  21. Herscovics A. Biosynthesis of thyroglobulin: incorporation of [3H] fucose into proteins by rat thyroids in vitro. Biochem J. 1970 Apr;117(2):411–413. doi: 10.1042/bj1170411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kondo Y., De Nayer P., Salabe G., Robbins J., Rall J. E. Function of isolated bovine thyroid polyribosomes. Endocrinology. 1968 Nov;83(5):1123–1131. doi: 10.1210/endo-83-5-1123. [DOI] [PubMed] [Google Scholar]
  23. Kopriwa B. M. A reliable, standardized method for ultrastructural electron microscopic radioautography. Histochemie. 1973 Oct 3;37(1):1–17. doi: 10.1007/BF00306855. [DOI] [PubMed] [Google Scholar]
  24. Kopriwa B. A comparison of various procedures for fine grain development in electron microscopic radioautography. Histochemistry. 1975 Aug 28;44(3):201–224. doi: 10.1007/BF00491492. [DOI] [PubMed] [Google Scholar]
  25. Kraehenbuhl J. P., Jamieson J. D. Localization of intracellular antigens by immunoelectron microscopy. Int Rev Exp Pathol. 1974;13(0):1–53. [PubMed] [Google Scholar]
  26. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  27. Leduc E. H., Bernhard W. Recent modifications of the glycol methacrylate embedding procedure. J Ultrastruct Res. 1967 Jul;19(1):196–199. doi: 10.1016/s0022-5320(67)80068-6. [DOI] [PubMed] [Google Scholar]
  28. METZGER H., SHARP G. C., EDELHOCH H. The properties of thyroglobulin. VII. The immunoloic activity of thyroglobulin fragments. Biochemistry. 1962 Mar;1:205–214. doi: 10.1021/bi00908a003. [DOI] [PubMed] [Google Scholar]
  29. McConahey P. J., Dixon F. J. A method of trace iodination of proteins for immunologic studies. Int Arch Allergy Appl Immunol. 1966;29(2):185–189. doi: 10.1159/000229699. [DOI] [PubMed] [Google Scholar]
  30. Morais R., Goldberg I. H. Cell-free synthesis of thyroglobulin. Biochemistry. 1967 Aug;6(8):2538–2545. doi: 10.1021/bi00860a035. [DOI] [PubMed] [Google Scholar]
  31. Moriarty G. C., Halmi N. S. Electron microscopic study of the adrenocorticotropin-producing cell with the use of unlabeled antibody and the soluble peroxidase-antiperoxidase complex. J Histochem Cytochem. 1972 Aug;20(8):590–603. doi: 10.1177/20.8.590. [DOI] [PubMed] [Google Scholar]
  32. Moriarty G. C., Moriarty C. M., Sternberger L. A. Ultrastructural immunocytochemistry with unlabeled antibodies and the peroxidase-antiperoxidase complex. A technique more sensitive than radioimmunoassay. J Histochem Cytochem. 1973 Sep;21(9):825–833. doi: 10.1177/21.9.825. [DOI] [PubMed] [Google Scholar]
  33. NADLER N. J., SARKAR S. K., LEBLOND C. P. Origin of intracellular colloid droplets in the rat thyroid. Endocrinology. 1962 Jul;71:120–129. doi: 10.1210/endo-71-1-120. [DOI] [PubMed] [Google Scholar]
  34. NADLER N. J., YOUNG B. A., LEBLOND C. P., MITMAKER B. ELABORATION OF THYROGLOBULIN IN THE THYROID FOLLICLE. Endocrinology. 1964 Mar;74:333–354. doi: 10.1210/endo-74-3-333. [DOI] [PubMed] [Google Scholar]
  35. NUNEZ J., MAUCHAMP J., MACCHIA V., JERUSALMI A., ROCHE J. BIOSYNTHESIS OF THYROGLOBULIN BY ISOLATED THYROID CELLS AND CELL-FREE SYSTEM. Biochem Biophys Res Commun. 1965 Jun 18;20:71–77. doi: 10.1016/0006-291x(65)90952-6. [DOI] [PubMed] [Google Scholar]
  36. Novikoff A. B., Novikoff P. M., Quintana N., Davis C. Diffusion artificats in 3,3'-diaminobenzidine cytochemistry. J Histochem Cytochem. 1972 Sep;20(9):745–749. doi: 10.1177/20.9.745. [DOI] [PubMed] [Google Scholar]
  37. Palade G. Intracellular aspects of the process of protein synthesis. Science. 1975 Aug 1;189(4200):347–358. doi: 10.1126/science.1096303. [DOI] [PubMed] [Google Scholar]
  38. Pelletier G., Puviani R., Dussault J. H. Electron microscope immunohistochemical localization of thyroglobulin in the rat thyroid gland. Endocrinology. 1976 May;98(5):1253–1259. doi: 10.1210/endo-98-5-1253. [DOI] [PubMed] [Google Scholar]
  39. Rolland M., Lissitzky S. Polypeptides non-covalently associated in 19-S thyroglobulin. Biochim Biophys Acta. 1972 Sep 29;278(2):316–336. doi: 10.1016/0005-2795(72)90237-1. [DOI] [PubMed] [Google Scholar]
  40. Rothman S. S. Protein transport by the pancreas. Science. 1975 Nov 21;190(4216):747–753. doi: 10.1126/science.1105785. [DOI] [PubMed] [Google Scholar]
  41. Salabe G., Kondo Y., DeNayer P., Robbins J., Rall J. E. Immunochemical identification of thyroglobulin fragments synthesized in vitro by bovine thyroid polyribosomes. Endocrinology. 1969 Oct;85(4):752–762. doi: 10.1210/endo-85-4-752. [DOI] [PubMed] [Google Scholar]
  42. Spiro M. J. Subunit heterogeneity of thyroglobulin. J Biol Chem. 1973 Jun 25;248(12):4446–4460. [PubMed] [Google Scholar]
  43. Spiro R. G., Spiro M. J. Glycoprotein biosynthesis: studies on thyroglobulin. Characterization of a particulate precursor and radioisotope incorporation by thyroid slices and particle systems. J Biol Chem. 1966 Mar 25;241(6):1271–1282. [PubMed] [Google Scholar]
  44. Sternberger L. A., Petrali J. P. Quantitative immunocytochemistry of pituitary receptors for luteinizing hormone-releasing hormone. Cell Tissue Res. 1975 Sep 17;162(2):141–176. doi: 10.1007/BF00209204. [DOI] [PubMed] [Google Scholar]
  45. Strum J. M., Karnovsky M. J. Cytochemical localization of endogenous peroxidase in thyroid follicular cells. J Cell Biol. 1970 Mar;44(3):655–666. doi: 10.1083/jcb.44.3.655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Tice L. W., Wollman S. H. Ultrastructural localization of peroxidase activity on some membranes of the typical thyroid epithelial cell. Lab Invest. 1972 Jan;26(1):63–73. [PubMed] [Google Scholar]
  47. WOLFF J. TRANSPORT OF IODIDE AND OTHER ANIONS IN THE THYROID GLAND. Physiol Rev. 1964 Jan;44:45–90. doi: 10.1152/physrev.1964.44.1.45. [DOI] [PubMed] [Google Scholar]
  48. Whur P., Herscovics A., Leblond C. P. Radioautographic visualization of the incorporation of galactose-3H and mannose-3H by rat thyroids in vitro in relation to the stages of thyroglobulin synthesis. J Cell Biol. 1969 Nov;43(2):289–311. doi: 10.1083/jcb.43.2.289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Zacharius R. M., Zell T. E., Morrison J. H., Woodlock J. J. Glycoprotein staining following electrophoresis on acrylamide gels. Anal Biochem. 1969 Jul;30(1):148–152. doi: 10.1016/0003-2697(69)90383-2. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES