Summary
The protein A-gold technique is amongst the most useful labeling techniques available for light and electron microscopic immunolabeling. Some electron microscopic studies, however, have suggested that protein A-gold, and other protein-gold complexes as well, may bind non-specifically to certain tissue structures, particularly in skin, creating a specious pattern of labeling.
We utilized the protein A-gold technique with antiserum to both involucrin and keratin under a variety of conditions to document the specificity of labeling. When the standard conditions were followed, the protein A-gold technique produces highly specific results. These conditions include: 1. the blocking of unreacted aldehyde groups by amination; 2. the blocking of non-specific binding sites on tissue sections by preincubation with inert proteins; and 3. the use of proper concentration of the protein A-gold complex. However, non-specific labeling could be produced if the three components of the standard protocol were omitted. In particular, the use of too concentrated protein A-gold lead to non-specific labeling.
We report here also updated working protocols for antigen detection with protein A-gold on semithin Lowicryl K4M and paraffin sections which provide optimal staining results.
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References
Asada-Kubota M (1988) Selective binding of colloidal gold-protein conjugates to secretory granules of granular skin glands and myelin sheaths in the skin of Xenopus laevis. J Ultrastruc Mol Struct Res 98:147–157
Behnke O, Ammitzbøll T, Jessen H, Klokker M, Nilausen K, Tranum-Jensen J, Olsson L (1986) Non-specific binding of protein-stabilized gold sols as a source of error in immunocytochemistry. Eur J Cell Biol 41:326–338
Bendayan M (1984) Protein A-gold electron microscopic immunocytochemistry: methods, applications, and limitations. J Electron Microsc Techn 1:243–270
Bendayan M, Nanci A, Kan FWK (1987) Effect of tissue processing on colloidal gold cytochemistry. J Histochem Cytochem 35:983–996
Brada D, Roth J (1984) “Golden blot”-detection of polyclonal and monoclonal antibodies bound to antigens on nitrocellulose by protein A-gold complex. Anal Biochem 142:79–83
Carlemalm E, Garavito M, Villiger W (1982) Resin development for electron microscopy and an analysis of embedding at low temperature. J Microsc 126:123–143
Frens G (1973) Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions. Nature (Phys Sci) 241:20–30
Frosch M, Görgen I, Boulnois GJ, Timmis KN, Bitter-Suermann D (1985) NZB mouse system for production of monoclonal antibodies to weak bacterial antigens: Isolation of an IgG antibody to the polysaccharide capsules of E. coli K1 and group B meningococci. Proc Natl Acad Sci USA 82:1194–1198
Geuze HJ, Slot JW, Scheffer RCT, van der Ley PA (1981) Use of colloidal gold particles in double-labeling immunoelectron microscopy of ultrathin frozen tissue sections. J Cell Biol 89:653–665
Holgate CS, Jackson PS, Cowen PN, Bird CC (1983) Immunogold-silver staining: a new method of immunostaining with enhanced sensitivity. J Histochem Cytochem 31:939–944
Jessen H, Behnke O (1986) Selective binding of colloidal gold-protein conjugates to epidermal phosphorus-rich keratohyaline granules and cornified cells. J Invest Dermatol 87:737–740
Langone JJ (1982) Protein A of staphylococcus aureus and related immunoglobulin receptors produced by streptococci and pneumococci. Adv Immunol 32:157–252
Lucocq JM, Roth J (1985) Application of immunocolloids in light microscopy. III. Demonstration of antigenic and lectin-binding sites in semithin resin sections. J Histochem Cytochem 32:1075–1085
Mau S-L, Clarke AE (1983) Binding of a plant glycoprotein to staphylococcal protein A. Phytochemistry 22:91–95
Moeremans M, Daneels G, Van Dijck A, Langanger G, De Mey J (1984) Sensitive visualization of antigen-antibody reactions in dot blot and blot immune overlay assays with immunogold and immunogold/silver staining. J Immunol Methods 74:353–560
Rice RH, Green H (1979) Presence in human epidermal cells of a soluble protein precursor of the cross-linked envelope: activation of the cross-linking by calcium ions. Cell 18:681–694
Roth J (1982a) The protein A-gold (pAg) technique-a qualitative and quantitative approach for antigen localization on thin sections. In: Bullock GR, Petrusz P (eds) Techniques in immunocytochemistry, vol 1. Academic Press, London, pp 107–133
Roth J (1982b) Applications of immunocolloids in light microscopy. Preparation of protein A-silver and protein A-gold complexes and their application for localization of single and multiple antigens in paraffin sections. J Histochem Cytochem 30:691–696
Roth J (1983) The colloidal gold marker system for light and electron microscopic cytochemistry. In: Bullock GR, Petrusz P (eds) Techniques in immunocytochemistry, vol 3. Academic Press, London, pp 217–284
Roth J (1986) Post-embedding cytochemistry with gold-labeled reagents: a review. J Microsc 14:123–137
Roth J (1987) Light and electron microscopic localization of glycoconjugates with gold-labeled reagents. Scanning Microsc 1:695–704
Roth J, Berger EG (1982) Immunocytochemical localization of galactosyltransferase in HeLa cells: codistribution with thiamine pyrophosphatase in trans Golgi cisternae. J Cell Biol 93:223–229
Roth J, Bendayan M, Orci L (1978) Ultrastructural localization of intracellular antigens by the use of protein A-gold complex. J Histochem Cytochem 26:1074–1081
Roth J, Bendayan M, Carlemalm E, Villiger W, Garavito M (1981a) Enhancement of structural preservation and immunocytochemical staining in low temperature-embedded pancreatic tissue. J Histochem Cytochem 29:663–671
Roth J, Thorens B, Hunziker W, Norman AW, Orci L (1981b) Vitamin D-dependent calcium binding protein: immunocytochemical localization in chick kidney. Science 214:197–200
Roth J, Lucocq JM, Charest PM (1984) Light and electron microscopic demonstration of sialic acid residues with the lectin from Limax flavus. A cytochemical affinity technique with the use of fetuin-gold complexes. J Histochem Cytochem 32:1167–1176
Roth J, Taatjes DJ, Bitter-Suermann D, Finne J (1987) Polysialic acid units are spatially and temporally expressed in developing postnatal rat kidney. Proc Natl Acad Sci USA 84:1969–1973
Roth J, Taatjes DJ, Wagner P, Weisgerber C, Heitz PhU, Goridis C, Bitter-Suermann D (1988) Reexpression of poly(sialic acid) units of the neural cell adhesion molecule in Wilms tumor. Proc Natl Acad Sci USA 85:2999–3003
Slot JW, Geuze HJ (1985) A new method of preparing gold probes for multiple-labeling cytochemistry. Eur J Cell Biol 38:87–93
Springall DR, Hacker GW, Grimelius L, Polak JM (1984) The potential of the immunogold-silver staining method, for paraffin sections. Histochemistry 81:603–608
Stathis EC, Fabrikanos A (1958) Preparation of colloidal gold. Chem Ind 27:860–861
Taatjes DJ, Roth J, Peumans W, Goldstein IJ (1988) Elderberry bark lectin-gold techniques for the detection of Neu5Ac (α2,6) Gal/GalNAc sequences: applications and limitations. Histochem J 20:478–490
Viac J, Reano A, Brochier J, Staquet M-J, Thivolet J (1983) Reactivity pattern of a monoclonal antikeratin antibody (KL 1). J Invest Dermatol 81:351–354
Warhol M, Roth J, Lucocq JM, Pinkus GS, Rice RH (1985a) Immuno-ultrastructural localization of involucrin in squamous epithelium and cultured keratinocytes. J Histochem Cytochem 33:141–149
Warhol MJ, Lucocq JM, Carlemalm E, Roth J (1985b) Ultrastructural localization of keratin proteins in human skin using low-temperature embedding and the protein A-gold technique. J Invest Dermatol 84:69–72
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Roth, J., Taatjes, D.J. & Warhol, M.J. Prevention of non-specific interactions of gold-labeled reagents on tissue sections. Histochemistry 92, 47–56 (1989). https://doi.org/10.1007/BF00495015
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DOI: https://doi.org/10.1007/BF00495015