Abstract
Cell biology concerns the interactions between different cellular compartments and between the cell and the environment. The mechanisms of herpes simplex virus type-1 (HSV-1) envelopment and the transport of virus particles and HSV-1 glycoproteins have not been completely investigated. It is of interest to examine the formation of complete virus particles and the cellular distribution of viral glycoproteins correlated with microtubules. The illustration of these conditions by immunocytochemistry is best done by multiple labelling techniques in the same cell. Single-staining of neighbouring serial sections or two-face double-immunolabelling methods are not technically compatible with ultrathin cryosections. The results are reported here of a simultaneous, simple and reliable immunogold double-staining technique using primary antibodies of the same species in ultrathin cryosections. Compared to other inactivation procedures, phosphate-buffered 3% paraformaldehyde plus 2% glutaraldehyde for 2 h at room temperature is an excellent and gentle method to destroy free anti-IgG binding sites on the antibodies and to prevent cross-labelling, which has proven necessary for obtaining reproducible results on cellular distribution of tubulin and viral glycoproteins gD-1 and gC-1.
Similar content being viewed by others
References cited
Balslev Y, Hansen GH (1989) Preparation and use of recombinant protein G-gold complexes as markers in double labelling immunocytochemistry. Histochem J 21: 449-454.
Baschong W, Lucocq JM, Roth J (1985) 'Thiocyanate gold': small (2-3 nm) colloidal gold for affinity cytochemical labeling in electron microscopy. Histochemistry 83: 409-411.
Bendayan M (1982) Double immunocytochemical labeling applying the protein A-gold technique. J Histochem Cytochem 30: 81-85.
Bendayan M, Nanci A, Herbener GH, Grégoire S, Duhr MA (1986) A review of the study of protein secretion applying the protein A-gold immunocytochemical approach. Am J Anat 175: 379-400.
Bienz K, Egger D, Pasamontes L (1986) Electron microscopic immunocytochemistry. Silver enhancement of colloidal gold marker allows double labeling with the same primary antibody. J Histochem Cytochem 34: 1337-1342.
Doerr-Schott J (1989) Colloidal gold for multiple staining. In: Hayat MA ed. Colloidal Gold. Principles, Methods, and Applications, Vol. 1. San Diego: Academic Press, pp. 145-190.
Edson CM, Hosler BA, Poodry CA, Schooley RT, Waters DJ, Thorley-Lawson DA (1985) Varicella-zoster virus envelope glycoproteins: biochemical characterization and identification in clinical material. Virology 145: 62-71.
Eisenberg RJ, Long D, Ponce de Leon M, Matthews JT, Spear PG, Gibson MG, Lasky LA, Berman P, Golub E, Cohen GH (1985) Localization of epitopes of herpes simplex virus type 1 glycoprotein D. J Virol 53: 634-644.
Ejercito PM, Kieff ED, Roizman B (1968) Characterization of herpes simplex virus strains differing in their effects on social behaviour of infected cells. J Gen Virol 2: 357-364.
Faulk WP, Taylor GM (1971) An immunocolloid method for the electron microscope. Immunochemistry 8: 1081-1083.
Gelderblom HR, Kocks C, L'age-Stehr J, Reupke H (1985) Comparative immunoelectron microscopy with monoclonal antibodies on yellow fever virus-infected cells: pre-embedding labelling versus immunocryoultramicrotomy. J Virol Methods 10: 225-239.
Gershon AA, Sherman DL, Zhu Z, Gabel CA, Ambron RT, Gershon MD (1994) Intracellular transport of newly synthesized varicella-zoster virus: final envelopment in the trans-golgi network. J Virol 68: 6372-6390.
Geuze HJ, Slot JW, Van der Ley PA, Scheffer RCT, Griffith JM (1981) Use of colloidal gold particles in double-labeling immunoelectron microscopy of ultrathin frozen tissue sections. J Cell Biol 89: 653-665.
Gong M, Kieff E (1990) Intracellular trafficking of two major Epstein-Barr virus glycoproteins, gp350/220 and gp110. JVirol 64: 1507-1516.
Görcs TJ, Léránth C, Maclusky NJ (1986) The use of gold-substituted silver-intensified diaminobenzidine (DAB) and non-intensified DAB for simultaneous electron microscopic immunoperoxidase labeling of tyrosine hydroxylase and glutamic acid decarboxylase immunoreactivity in the rat medial preoptic area. J Histochem Cytochem 34: 1439-1447.
Grøndahl-Hansen J, Ralfkiær E, Nielsen LS, Kristensen P, Frentz G, Danø, K (1987) Immunohistochemical localization of urokinase-and tissue-type plasminogen activators in psoriatic skin. J Invest Dermatol 88: 28-32.
Hari V, Baunoch D, Das P (1990) Sphere-linked immunodiagnostic assay (SLIDA): an electron microscopic method for detecting specific antibodies. Bio Techniques 9: 342-350.
Horisberger M (1983) Colloidal gold as a tool in molecular biology. TIBS November: 395-397.
Isola VJ, Eisenberg RJ, Siebert GR, Heilman CJ, Wilcox WC, Cohen GH (1989) Fine mapping of antigenic site II of herpes simplex virus glycoprotein D. J Virol 63: 2325-2334.
Jensen HL, Norrild B (1998) Herpes simplex virus type 1-infected human embryonic lung cells studied by optimized immunogold cryosection electron microscopy. J Histochem Cytochem 46: 487-496.
Jensen H, Broholm N, Norrild B (1995) Low-fading immunofluorescence with propidium iodide contrast compared with immunogold light microscopy in whole cells and semi-thin cryosections. J Histochem Cytochem 43: 507-513.
Koga J, Chatterjee S, Whitley RJ (1986) Studies on herpes simplex virus type 1 glycoproteins using monoclonal antibodies. Virology 151: 385-389.
Komuro M, Tajima M, Kato K (1989) Transformation of Golgi membrane into the envelope of herpes simplex virus in rat anterior pituitary cells. Eur J Cell Biol 50: 398-406.
Larsson L-I (1988) Immunocytochemistry: Theory and Practice. Boca Raton, FL: CRC Press.
Larsson L-I (1979) Simultaneous ultrastructural demonstration of multiple peptides in endocrine cells by a novel immunocytochemical method. Nature 282: 743-746.
La Thangue NB, Chan WL, Almeida JD (1984) Monoclonal antibodies to herpes simplex virus type 1 glycoproteins show that epitope location influences virus neutralization. J Med Virol 13: 227-242.
Lausch RN, Staats H, Oakes JE, Cohen GH, Eisenberg RJ (1991) Prevention of herpes keratitis by monoclonal antibodies specific for discontinuous and continuous epitopes on glycoprotein D. Invest Ophthalmol Vis Sci 32: 2735-2740.
Lopez-Iglesias C, Puvion-Dutilleul F (1988a) Ultrastructural localization of glycoproteins in rabbit fibroblasts altered by herpes simplex virus type 1 infection. Biol Cell 62: 47-56.
Lopez-Iglesias C, Puvion-Dutilleul F (1988b) Visualization of glycoproteins after tunicamycin and monensin treatment of herpes simplex virus infected cells. J Ultrastructure Mol Structure Res 101: 75-91.
Nielsen MH, Bastholm L, Chatterjee S, Koga J, Norrild B (1989) Simultaneous triple-immunogold staining of virus and host cell antigens with monoclonal antibodies of virus and host cell antigens in ultrathin cryosections. Histochemistry 92: 89-93.
Nii S, Morgan C, Rose HM, Hsu KC (1968) Electron microscopy of herpes simplex virus IV. Studies with ferritin-conjugated antibodies. J Virol 2: 1172-1184.
Norrild B, Lehto V-P, Virtanen I (1986) Organization of cytoskeleton elements during herpes simplex virus type 1 infection of human fibroblasts: an immunofluorescence study. J Gen Virol 67: 97-105.
Norrild B, Nielsen MH, Bastholm L, Chatterjee S (1991) Intracellular maturation and sorting of two herpes simplex virus type 1 glycoproteins. Immunogold staining of ultrathin cryosections. APMIS 99: 371-380.
Patterson S, Gross J, Oxford JS (1988) The intracellular distribution of influenza virus matrix protein and nucleoprotein in infected cells and their relationship to haemagglutinin in the plasma membrane. J Gen Virol 69: 1859-1872.
Pereira L, Klassen T, Baringer JR (1980) Type-common and type-specific monoclonal antibody to herpes simplex virus type 1. Infect Immun 29: 724-732.
Roth J, Binder M (1978) Colloidal gold, ferritin and peroxidase as markers for electron microscopic double labeling lectin techniques. J Histochem Cytochem 26: 163-169.
Scopsi L, Bock E, Larsson L-I (1986) Monoclonal antibody immunocytochemistry: novel method extending usefulness of monoclonal antibodies for antigen visualization. Eur J Cell Biol 41: 97-101.
Slot JW, Geuze HJ (1985) A new method of preparing gold probes for multiple-labeling cytochemistry. Eur J Cell Biol 38: 87-93.
Stannard LM, Fuller AO, Spear PG (1987) Herpes simplex virus glycoproteins associated with different morphological entities projecting from the virion envelope. J Gen Virol 68: 715-725.
Thorpe JR (1992) A novel methodology for double protein A-gold immunolabeling utilizing the monovalent fragment of protein A. J Histochem Cytochem 40: 435-441.
Van Bergen en Henegouwen PMP (1989) Immunogold labeling of ultrathin cryosections. In: Hayat MA ed. Colloidal Gold. Principles, Methods, and Applications, Vol. 1, San Diego: Academic Press, pp. 191-216.
Van den Pol AN (1985) Silver-intensified gold and peroxidase as dual ultrastructural immunolabels for pre-and postsynaptic neurotransmitters. Science 228: 332-335.
Vernon SK, Ponce de Leon M, Cohen GH, Eisenberg RJ, Rubin BA (1981) Morphological components of herpesvirus. III. Localization of herpes simplex virus type 1 nucleocapsid polypeptides by immune electron microscopy. J Gen Virol 54: 39-46.
Wang B-L, Larsson L-I (1985) Simultaneous demonstration of multiple antigens by indirect immunofluorescence or immunogold staining. Novel light and electron microscopical double and triple staining method employing primary antibodies from the same species. Histochemistry 83: 47-56.
Ward PL, Campadelli-Fiume G, Avitabile E, Roizman B (1994) Localization and putative function of the UL20 membrane protein in cells infected with herpes simplex virus 1. J Virol 68: 7406-7417.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Jensen, H.L., Norrild, B. Easy and Reliable Double-Immunogold Labelling of Herpes Simplex Virus Type-1 Infected Cells Using Primary Monoclonal Antibodies and Studied by Cryosection Electron Microscopy. Histochem J 31, 525–533 (1999). https://doi.org/10.1023/A:1003840006848
Issue Date:
DOI: https://doi.org/10.1023/A:1003840006848