Summary
Acridine Orange in concentrations from 0.01% to 0.2% was added to the first fixative solution in order to stain vibratome sections and small blocks of the articular cartilage of 2 month old rats. The interterritorial matrix of the radial or deep zone (zone 3) was examined. It contained reaction products with different morphology depending on the specimens used. In vibratome sections filaments were seen arranged in a homogenous pattern and changing in size with the concentration of the dye: diluted solutions produced finer filaments than concentrated ones. In contrast, in tissue blocks the staining pattern was not altered by different concentrations of Acridine Orange. However, with increase of the distance from the surface of the specimens the size of the filaments gradually decreased and formed a finer network. Since after preincubation with chondroitin ABC lyase only minute reaction products remained, an interaction of the dye with the sulphated glycosaminoglycans of the proteoglycans in the articular cartilage is suggested.
The experiments show that by using mainly monocationic monomers of Acridine Orange the proteoglycans can be stained in a more expanded state than with polycationic dye polymers.
Similar content being viewed by others
References
Bradley, D. F. &Wolf, M. K. (1959) Aggregation of dyes bound to polyanions.Proc. Natl. Acad. Sci. USA.45, 944–52.
Brandes, G. (1986)Proteoglykane in der glomerulären Basalmembran und in der hyalinen Knorpelmatrix. Elekronenmikroskopische Darstellung mit Hilfe von Akridinorange. Diss. Hannover.
Engfeldt, B. &Hjertquist, S.-O. (1967) The effect of various fixatives on the preservation of acid glycosaminoglycans in tissues.Acta Pathol. Microbiol. Scand. 71, 219–32.
Engfeldt, B. &Hjertquist, S.-O. (1968) Studies on the epiphysial growth zone. I. The preservation of acid glycosaminoglycans in tissues in some histochnical procedures for electron microscopy.Virchows Arch. Abt. B Zellpath. 1, 222–9.
Enzyme nomenclature (1984). Orlando: Academic Press, Inc.
Ghadially, F. N. (1983)Fine Structure of Synovial Joints. A Text and Atlas of the Ultrastructure of Normal and Pathological Articular Tissues. pp. 42–79. London: Butterworths.
Harms, H. (1965)Handbuch der Farbstoffe für die Mikroskopie. Kamp-Lintfort: Staufen Verlag.
Hascall, G. K. (1980) Cartilage proteoglycans: Comparison of sectioned and spread whole molecules.J. Ultrastruct. Res. 70, 369–75.
Hunziker, E. B., Herrmann, W. &Schenk, R. K. (1982) Improved cartilage fixation by ruthenium hexamine trichloride (RHT). A prerequisite for morphometry in growth cartilage.J. Ultrastruct. Res. 81, 1–12.
Hunzinker, E. B. &Schenk, R. K. (1984) Cartilage ultrastructure after high pressure freezing, freeze substitution, and low temperature embedding. II. Intercellular matrix ultrastructure—preservation of proteoglycans in their native state.J. Cell Biol. 98, 277–82.
Jozsa, L. &Szederkenyi, G. (1967) Über Verluste der Gewebsmukopolysaccharide während der Fixierung.Acta Histochem. 26, 255–60.
Kelly, J. W., Bloom, G. D. &Scott, J. E. (1963) Quarternary ammonium compounds in connective tissue histochemistry: I. Selective unblocking.J. Histochem. Cytochem. 11, 791–8.
Khan, T. A. &Overton, J. (1970) Lanthanum staining of developing chick cartilage and reaggregating cartilage cells.J. Cell Biol. 44, 433–8.
Kleine, T. O. (1981) Biosynthesis of proteoglycans: An approach to locate it in different membrane systems.Int. Rev. Conn. Tiss.Res. 9, 27–98.
Luft, J. H. (1971) Ruthenium red and violet. II. Fine structural localization in animal tissues.Anat. Rec. 171, 369–416.
Meachim, G. &Stockwell, R. A. (1979) The matrix. In:Adult Articular Cartilage (edited by Freeman, M. A. R.). 2nd edn. pp. 1–67. Tunbridge Wells: Pitman Medical Publishing Co. Ltd.
Michaelis, I. (1947) The nature of the interaction of nucleic acids and nuclei with basic dyestuffs.Cold Spring Harb. Symp. Quant. Biol. 12, 131–42.
Reale, E., Luciano, L. &Kühn, K.-W. (1983) Ultrastructural architecture of proteoglycans in the glomerular basement membrane. A cytochemical approach.J. Histochem. Cytochem. 31, 662–8.
Rosenberg, L., Hellmann, W. &Kleinschmidt, A.K. (1975) Electron microscopic studies of proteoglycan aggregates from bovine articular cartilage.J. Biol. Chem. 250, 1877–83.
Ruggeri, A., Dell'orbo, C. &Quacci, D. (1975) Electron microscopic visualization of proteoglycans with Alcian Blue.Histochem. J. 7, 187–97.
Ruggeri, A., Dell'orbo, C. &Quacci, D. (1977) Electron microscopic visualization of proteoglycans with Ruthenium Red.Histochem. J. 9, 249–52.
Schofield, B. H., Williams, B. R. &Doty, S. B. (1975) Alcian Blue staining of cartilage for electron microscopy. Application of the critical electrolyte concentration principle.Histochem. J. 7, 139–49.
Scott, J. E. (1980) Collagen—proteoglycan interactions. Localization of proteoglycans in tendon by electron microscopy.Biochem. J. 187, 887–91.
Scott, J. E. (1985) Proteoglycan histochemistry—a valuable tool for connective tissue biochemists.Collagen Rel. Res. 5, 541–75.
Scott, J. E. &Orford, C. R. (1981) Dermatan sulphate—rich proteoglycan associates with rat tail—tendon collagen at the d band in the gap region.Biochem. J. 197, 213–6.
Shepard, N. &Mitchell, N. (1976a) Simultaneous localization of proteoglycan by light and electron microscopy using toluidine blue O. A study of epiphyseal cartilage.J. Histochem. Cytochem. 24, 621–9.
Shepard, N. &Mitchell, N. (1976b) The localization of proteoglycan by light and electron microscopy using safranin 0. A study of epiphyseal cartilage.J. Ultrastruct. Res. 54, 451–60.
Shepard, N. &Mitchell, N. (1977) The localization of articular cartilage proteoglycan by electron microscopy.Anat. Rec. 187, 463–75.
Strugger, S. (1940) Fluoreszenzmikroskopische Untersuchungen über die Aufnahme und Speicherung des Akridinorange durch lebende und tote Pflanzenzellen.Jenaische Z. Med. Naturwiss. 73, 97–134.
Sylven, B. (1954) Metachromatic dye—substrate interactions.Quart. J. Microsc. Sci. 95, 327–58.
Szirmai, J. A. (1963) Quantitative approaches in the histochemistry of mucopolysaccharides.J. Histochem. Cytochem. 11, 24–34.
Takagi, M., Parmley, R. T., Toda, Y. &Austin, R. L. (1982) Ultrastructural cytochemistry and immunocytochemistry of sulfated glycosaminoglycans in epiphyseal cartilage.J. Histochem. Cytochem. 30, 1179–85.
Thyberg, J. (1977) Electron microscopy of cartilage proteoglycans.Histochem. J. 9, 259–66.
Thyberg, J., Lohmander, S. &Friberg, U. (1973) Electron microscopic demonstration of proteoglycans in guinea pig epiphyseal cartilage.J. Ultrastruct. Res. 45, 407–27.
Yamagata, T., Saito, H., Habuchi, O. &Suzuki, S. (1968) Purification and properties of bacterial chondroitinases and chondrosulfatases.J. Biol. Chem. 243, 1523–35.
Zanker, V. (1952a) Über den Nachweis definierter reversibler Assoziate (“reversible Polymerisate”) des Acridinorange durch Absorptions—und Fluoreszenzmessungen in wäßriger Lösung.Z. Phys. Chem. 199, 225–58.
Zanker, V. (1952b) Quantitative Absorptions- und Emissionsmessungen am Acridinorangekation bei Normal-und Tieftemperatur im organischen Lösungsmittel und ihr Beitrag zur Deutung des metachromatischen Fluoreszenzproblems.Z. Phys. Chem. 200, 250–92.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Brandes, G., Reale, E. The reaction of Acridine Orange with proteoglycans in the articular cartilage of the rat. Histochem J 22, 106–112 (1990). https://doi.org/10.1007/BF01885789
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF01885789