Summary
We studied the interaction between glycosaminoglycans (GAGs) and fibronectin in the basement membrane of the epiblast in the chicken blastoderm using testicular-hyaluronidase digestion of GAGs either on fixed tissue sections or in vivo after microinjection of the enzyme preparation prior to immunostaining for fibronectin. In the choice of fixatives, special attention was paid to their preservation of GAGs. The controls included alcian-blue staining of serial sections to test the efficiency of the digestion, and incubations in the presence of protease inhibitors to abolish contaminating proteolytic activity in the commercial hyaluronidase preparations. The results indicate that fixation in solutions which preserve GAGs, i.e. ethanolic solutions or aqueous solutions containing cetylpyridinium chloride, allows the immunocytochemical demonstration of fibronectin in the basement membrane of the epiblast at the level of the endophyllic crescent, but masks this glycoprotein at the epithelial-mesenchymal interface. As shown by both approaches, this masking of immunoreactivity is reversible. Moreover, the in vivo clearance of GAGs before fixation shows that the masking at the epithelial-mesenchymal interface is not an experimental peculiarity due to the use of a particular technique, but is the consequence of an interaction between GAGs and fibronectin in that particular area of the basement membrane that is used by mesoblast cells as a substrate for migration. The observation that fibronectin may be masked by GAGs in ethanol-fixed tissue — a commonly used fixation method—may require the re-evaluation of some negative results mentioned in the literature.
Similar content being viewed by others
References
Arnold W, Kalden JR, von Mayersbach H (1975) Influence of different histologic preparation methods on preservation of tissue antigens in the immunofluorescent antibody technique. Ann NY Acad Sci 254:27–34
Bergroth V, Reitamo S, Konttinen YT, Tolvanen E (1982) Fixation-dependent cytoplasmic false-positive staining with an immunoperoxidase method. Histochemistry 73:509–513
Clemmensen I, Hølund B, Johansen N, Andersen RB (1982) Demonstration of fibronectin in human articular cartilage by an indirect immunoperoxidase technique. Histochemistry 76:51–56
Dawson IMP (1972) Fixation: what should the pathologist do? Histochem J 4:381–385
Dessau W, Sasse J, Timpl R, Jilek F, von der Mark K (1978) Synthesis and extracellular deposition of fibronectin in chondrocyte cultures. J Cell Biol 79:342–355
Feltkamp-Vroom TM (1975) Preparation of tissues and cells for immunohistochemical processing. Ann NY Acad Sci 254:21–26
Fisher M, Solursh M (1977) Glycosaminoglycan localization and role in maintenance of tissue spaces in the early chick embryo. J Embryol Exp Morphol 42:195–207
Foidart J-M, Berman JJ, Paglia L, Rennard S, Abe S, Perantoni A, Martin GR (1980) Synthesis of fibronectin, laminin, and several collagens by a liver-derived epithelial cell line. Lab Invest 42:525–532
Harrisson F, Vanroelen Ch, Foidart J-M, Vakaet L (1984) Expression of different regional patterns of fibronectin immunoreactivity during mesoblast formation in the chick blastoderm. Dev Biol 101:373–381
Hølund B, Clemmensen I (1982) The value of hyaluronidase treatment of different tissues before demonstration of fibronectin by the indirect immunoperoxidase technique. Histochemistry 76:517–526
Hølund B, Clausen PP, Clemmensen I (1981) The influence of fixation and tissue preparation on the immunohistochemical demonstration of fibronectin in human tissue. Histochemistry 72:291–299
Hølund B, Clemmensen I, Wanning M (1984) Sequential appearance of fibronectin and collagen fibres in experimental arthritis in rabbits. Histochemistry 80:39–44
Hopwood D (1969) Fixatives and fixation: a review. Histochem J 1:323–360
Isemura M, Yasaziwa Z, Koide T, Ono T (1982) Interaction of fibronectin and its proteolytic fragments with hyaluronic acid. J Biochem (Tokyo) 91:731–734
Judd MA, Britten KJM (1982) Tissue preparation for the demonstration of surface antigens by immunoperoxidase techniques. Histochem J 14:747–754
Keiser HD, Hatcher VB (1977) A comparison of bovine nasal cartilage proteoglycan core protein produced by chondroitinase and hyaluronidase: the possible role of protease contaminants. Connect Tissue Res 5:147–155
Kosher RA, Walker KH, Ledger PW (1982) Temporal and spatial distribution of fibronectin during development of the embryonic chick limb bud. Cell Differ 11:217–228
Kleinman HK (1982) Interactions between connective tissue matrix macromolecules. Connect Tissue Res 10:61–72
Kuusela P, Ruoslahti E, Engvall E, Vaheri A (1976) Immunological interspecies cross-reactions of fibroblast surface antigen (fibronectin). Immunochemistry 13:639–642
Laterra J, Culp LA (1982) Differences in hyaluronate binding to plasma and cell surface fibronectins. J Biol Chem 257:719–726
Leppi TJ, Stoward PJ (1965) On the use of testicular hyaluronidase for identifying acid mucins in tissue sections. J Histochem Cytochem 13:406–407
Manasek FJ (1975) The extracellular matrix: a dynamic component of the developing embryo. In: Moscona AA, Monroy A (eds) Current topics in developmental biology, vol. 10. Academic Press, New York, pp 35–102
Mayer BW, Hay ED, Hynes RO (1981) Immunocytochemical localization of fibronectin in embryonic chick trunk and area vasculosa. Dev Biol 82:267–286
Melnick M, Jaskoll T, Brownell AG, MacDougall M, Bessem C, Slavkin HC (1981) Spatiotemporal patterns of fibronectin distribution during embryonic development. I. Chick limbs. J Embryol Exp Morphol 63:193–206
Mepham BL (1982) Influence of fixatives on the immunoreactivity of paraffin sections. Histochem J 14:731–738
Miller HRP (1972) Fixation and tissue preservation for antibody studies. Histochem J 4:305–320
New DAT (1955) A new technique for the cultivation of the chick embryo in vitro. J Embryol Exp Morphol 3:320–331
Oike Y, Kimata K, Shinomura T, Nakazawa K, Suzuki S (1980) Structural analysis of chick-embryo cartilage proteoglycan by selective degradation with chondroitin lyases (chondroitinases) and endo-β-D-galactosidase (keratanase). Biochem J 191:193–207
Perkins ME, Ji TH, Hynes RO (1979) Cross-linking of fibronectin to sulfated proteoglycans at the cell surface. Cell 16:941–952
Quintarelli G, Scott JE, Dellovo MC (1964) The chemical and histochemical properties of alcian blue. II. Dye binding of tissue polyanions. Histochemistry 4:86–98
Rennard SI, Berg R, Martin GR, Foidart J-M, Gehron Robey P (1980) Enzyme-linked immunoassay (ELISA) for connective tissue components. Anal Biochem 104:205–214
Sabatini DD, Bensch K, Barrnett RJ (1963) Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J Cell Biol 17:19–58
Sainte-Marie G (1962) A paraffin embedding technique for studies employing immunofluorescence. J Histochem Cytochem 10:250–256
Sanders EJ (1979) Development of the basal lamina and extracellular materials in the early chick embryo. Cell Tissue Res 198:527–537
Schenk E (1981) A newly certified dye: Alcian blue 8GX. Stain Technol 56:129–131
Scott JE (1955) The solubility of cetylpyridinium complexes of biological polyanions in solution of salts. Biochim Biophys Acta 18:428–429
Solursh M (1976) Glycosaminoglycan synthesis in the chick gastrula. Dev Biol 50:525–530
Szendröi M, Labat-Robert J, Godeau G, Robert AM (1983) Immunohistochemical detection of fibronectin using different fixatives in paraffin embedded sections. Pathol Biol 31:631–636
Vakaet L (1970) Cinephotomicrographic investigations of gastrulation in the chick blastoderm. Arch Biol (Liège) 81:387–426
Van Ewijk W, Coffman RC, Weissman IL (1980) Immunoelectron microscopy of cell surface antigens: a quantitative analysis of antibody binding after different fixation protocols. Histochem J 12:349–361
Van Ewijk W, Van Soest PL, Verkerk A, Jongkind JF (1984) Loss of antibody binding to prefixed cells: fixation parameters for immunocytochemistry. Histochem J 16:179–194
Van Hoof J, Harrisson F, Vakaet L (1984) Microinjection in the chick blastoderm: An improved method to study the extracellular matrix in the living organism. Exp Cell Res 155:278–282
Vanroelen Ch, Vakaet LCA (1981) Incorporation of35S-sulphate in chick blastoderms during elongation and during shortening of the primitive streak. Wilhelm Roux's Arch 190:233–236
Vanroelen Ch, Vakaet L, Andries L (1980a) Localization and characterization of acid mucopolysaccharides in the early chick blastoderm. J Embryol Exp Morphol 56:169–178
Vanroelen Ch, Vakaet L, Andries L (1980b) Distribution and turnover of testicular hyaluronidase sensitive macromolecules in the primitive streak stage chick blastoderm as revealed by autoradiography. Anat Embryol 159:361–367
Vanroelen Ch, Vakaet L, Andries L (1980c) Alcian blue staining during the formation of mesoblast in the primitive streak stage chick blastoderm. Anat Embryol 160:361–367
Wakely J, England MA (1979) Scanning electron microscopical and histochemical study of the structure and function of basement membranes in the early chick embryo. Proc R Soc Lond B 206:329–352
Weiss RE, Reddi AH (1980a) Immunofluorescent localization of fibronectin during endochondral bone formation. Fed Proc 39:956
Weiss RA, Reddi AH (1980b) Synthesis and localization of fibronectin during collagenous matrix-mesenchymal cell interaction and differentiation of cartilage and bone in vivo. Proc Natl Acad Sci USA 77:2074–2078
Weiss RE, Reddi AH (1981) Appearance of fibronectin during the differentiation of cartilage, bone, and bone marrow. J Cell Biol 88:630–636
Yamada KM (1978) Immunological characterization of a major transformation-sensitive fibroblast cell surface glycoprotein. Localization, redistribution, and role in cell shape. J Cell Biol 78:520–541
Yamada KM, Kennedy DW, Kimata K, Pratt RM (1980) Characterization of fibronectin interactions with glycosaminoglycans and identification of active proteolytic fragments. J Biol Chem 255:6055–6063
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Harrisson, F., Van Hoof, J., Vanroelen, C. et al. Masking of antigenic sites of fibronectin by glycosaminoglycans in ethanol-fixed embryonic tissue. Histochemistry 82, 169–174 (1985). https://doi.org/10.1007/BF00708202
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00708202