Electron Microscopic Preembedding Double-Immunostaining Methods

  • Csaba Leranth
  • Virginia M. Pickel


During the last decade, immunocytochemistry has become one of the major tools for the light and electron microscopic identification of neurons containing classical transmitters and neuropeptides (e.g., Cuello, 1983; Polak and Van Noorden, 1983; Steinbush et al., 1978). The immunocytochemical studies have provided a complete characterization of the regional distributions and ultrastructural morphology of a variety of chemically specific neurons (Elde etat., 1976; Pickel, 1981). More recently, dual immunocytochemical labeling of two antigens in the same section prepared for electron microscopy has permitted the characterization of transmitters within pre- and post synaptic junctions in the central nervous system (Leranth et al., 1984; van den Pol, 1985a; Milner et al., 1987).


Tyrosine Hydroxylase Colloidal Gold Glutamic Acid Decarboxylase Primary Antiserum Double IMMUNOSTAINING 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Abdel-Akher, M., Hamilton, J. K., Montgomery, R., and Smith, F., 1952, A new procedure for the demonstration of the fine structure of polysacharide, J. Am. Chem. Soc. 74:4970–4971.CrossRefGoogle Scholar
  2. Aimsworth, S. K., and Karnovsky, M. J., 1972, An ultrastructural staining method for enhancing the size and electron opacity of ferritin in the sections, J. Histochem. Cytochem. 20:225–229.CrossRefGoogle Scholar
  3. Beaudet, A., 1982, High resolution radioautography of central 5-hydroxytryptamine (5-HT) neurons, J. Histochem Cytochem. 9:765–768.CrossRefGoogle Scholar
  4. Bendayan, M., 1982, Double immunocytochemical labeling applying to protein A-gold technique, J. Histochem. Cytochem. 30:81–85.PubMedCrossRefGoogle Scholar
  5. Berod, A., Hartman, B. K., and Pujol, J. F., 1981, Importance of fixation in immunohistochem-istry: use of formaldehyde solutions at variable pH for the localization of thyrosine hydroxylase, J. Histochem. Cytochem. 29:844–850.PubMedCrossRefGoogle Scholar
  6. Bolam, J. P., Ingham, C. A., Izzo, P. N., Levey, A. I., Rye, D. B., Smith, A. D., and Wainer, B. H., 1986, Substance P-containing terminals in synaptic contact with cholinergic neurons in the neostriatum and basal forebrain: A double immunocytochemical study in the rat, Brain Res. 397:279–289.PubMedCrossRefGoogle Scholar
  7. Brunk, U., Brun, A., and Skold, G., 1966, Histochemical demonstration of heavy metals with the sulphide-silver method. Methodological study, Acta Histochem. 31:345–357.Google Scholar
  8. Cuello, A. C. (ed.), 1983, IBRO Handbook Series: Methods in Neurosciences, Volume 3, Immunohis-tochemistry, John Wiley & Sons, Chichester.Google Scholar
  9. Cuello, A. C., Priestley, J. V., and Milstein, C., 1982, Immunocytochemistry with internally labeled monoclonal antibodies, Proc. Natl. Acad. Sci. U.S.A. 79:665–669.PubMedCrossRefGoogle Scholar
  10. Danscher, G., 1981, Histochemical demonstration of heavy metals. A revised version of the sul-phid silver method suitable for both light and electron microscopy, Histochemistry 71:1–16.PubMedCrossRefGoogle Scholar
  11. DeMey, J., 1983, A critical review of light and electron microscopic immunocytochemical techniques used in neurobiology, J. Neurosci. Methods 7:1–18.CrossRefGoogle Scholar
  12. DeMey, J., Moermans, M., Guens, G. Nuydens, R., and DeBrabender, M., 1981, High-resolution light and electron microscopic localization of tubulin with the IGS (immuno gold staining) method, Biol. Int. Rep. 5:889–899.CrossRefGoogle Scholar
  13. Descaries, L., and Beaudet, A., 1983, Use of radioautography for investigation of transmitter-specific neurons, in: Handbook of Chemical Neuroanatomy, Volume 3 (A. Bjorklund and T. Hokfelt, eds.), Elsevier, Amsterdam, pp. 286–364.Google Scholar
  14. Doerr-Schott, J., and Garaud, J. C., 1981, Ultrastructural identification of gastrin-like immu-noreactive nerve fibers in the brain of Xenopus laevis by means of colloidal gold or ferritin immunocytochemical methods, Cell Tissue Res. 216:581–589.PubMedCrossRefGoogle Scholar
  15. Eckenstein, F., and Theonen, H., 1982, Production of specific antisera and monoclonal antibodies to choline acetyltransferase: Characterization and use for identification of cholinergic neurons, EMBOJ. 1:363–368.Google Scholar
  16. Eide, R., Hokfelt, T., Johansson, O., and Terenius, L., 1976, Immunohistochemical studies using antibodies to leucine-enkephalin: Initial observations of the nervous system of the rat, Neuroscience 1:349–351.CrossRefGoogle Scholar
  17. Faulk, V. P., and Taylor, G. P., 1971, An immunocolloid method for electron microscope, Immunochemistry 8:1081–1083.PubMedCrossRefGoogle Scholar
  18. Ferns, G., 1973, Controlled nucleation for the regulation of the particle size in nondisperse gold solutions, Nature 241:20–22.Google Scholar
  19. Fertuck, H. C., and Salpeter, M. M., 1974, Sensitivity in electron microscopic autoradiography for 125I, J. Histochem. Cytochem. 22:80–87.PubMedCrossRefGoogle Scholar
  20. Friedrich, V. L., and Mugnaini, E., 1981, Electron microscopy: Preparation of neural tissues for electron microscopy, in: Neuroanatomical Tract-Tracing Methods (L. Heimer and M. J. Robards, eds.), Plenum Press, New York, pp. 345–377.CrossRefGoogle Scholar
  21. Gallyas, F., 1971, A principle for silver staining of tissue elements by physical development, Acta Morphol. Acad. Sci. Hung. 19:57–71.PubMedGoogle Scholar
  22. Gallyas, F., 1982, Suppression of argyrophil III reaction by mercaptocompounds. Prerequisite for intensification of histochemical reactions by physical developers, Acta Histochem. 70:99–105.PubMedCrossRefGoogle Scholar
  23. Glazer, E. J., Ramachandran, J., and Basbaum, A. I., 1984, Radioimmunocytochemistry using a tritiated goat anti-rabbit second antibody, J. Histochem. Cytochem. 32:778–782.PubMedCrossRefGoogle Scholar
  24. Göres, T., Leranth, C., and MacLusky, N.J., 1986, The use of gold substituted silver-intensified diaminobensidine (DAB) and non-intensified DAB for simultaneous electron microscopic labeling of tyrosine hydroxylase and glutamic acid decarboxylase immunoreactivity in the rat medial preoptic area, J. Histochem. Cytochem. 34:1439–1447.CrossRefGoogle Scholar
  25. Graham, R. C., Jr., and Karnovsky, M. J., 1966, The early stages of a absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: Ultrastructural cytochemistry by a new technique, J. Histochem. Cytochem. 14:291–302.PubMedCrossRefGoogle Scholar
  26. Haug, F. M., 1967, Electron microscopic localization of the zinc in hippocampal mossy fibre synapses by a modified sulfide procedure, Histochemie 8:355–368.PubMedCrossRefGoogle Scholar
  27. Horisberger, M., 1982, Evaluation of colloidal gold as a cytochemical marker for transmission and scanning electron microscopy, Biol. Cell. 36:253–258.Google Scholar
  28. Horisberger, M., and Rosset, J., 1977, Colloidal gold, a useful marker for transmission and scanning electron microscopy, J. Histochem. Cytochem. 25:295–305.PubMedCrossRefGoogle Scholar
  29. Horisberger, M., Farr, D. R., and Vonlanthen, M., 1978, Ultrastructural localization of D-gal-actan in the nuclei of the myxomycete(Physarum polycephalum), Biochim. Biophys. Acta 542:308–314.CrossRefGoogle Scholar
  30. Hsu, S. M., Raine, L., and Fayer, H., 1981, The use of avidin—biotin-peroxidase complex (ABC) in immunoperoxidase technique: A comparison between ABC and unlabeled antibody (peroxidase) procedures, J. Histochem. Cytochem. 29:577–590.PubMedCrossRefGoogle Scholar
  31. Hudgson, A. J., Penke, B., Erdei, A., Chubb, I. W., and Somogyi, P., 1985, Antisera to gamma amino butyric acid. I. Production and characterization using a new model system, J. Histochem. Cytochem. 33:229–239.CrossRefGoogle Scholar
  32. Hunt, S. P., and Mantyh, P. W., 1984, Radioimmunohistochemistry with 3H-biotin, Brain Res., 291:203–217.PubMedCrossRefGoogle Scholar
  33. Joh, T. H., Gegham, C., an Reis, D. J., 1973, Immunochemical demonstration of increased tyrosine hydroxylase protein in sympathetic ganglia and adrenal medulla elicited by reser-pine, Proc. Natl. Acad. Sci. U.S.A. 70:2767–2771.PubMedCrossRefGoogle Scholar
  34. King, J. C., Lechan, R. M., Kugel, G., and Anthony, E. L. P., 1983, Acrolein: A fixative for immunocytochemical localization of peptides in the central nervous system, J. Histochem. Cytochem. 31:62–68.PubMedCrossRefGoogle Scholar
  35. Kohno, J., Shinoda, K., Kawai, Y., Ohuchi, T., Ono, K., and Shiotani, Y., 1988, Interaction between adrenergic fibers and intermediate cholinergic neurons in the rat spinal cord: A new double-immunostaining method for correlated light and electron microscopic observations, Neuroscience 25:113–121.PubMedCrossRefGoogle Scholar
  36. Lakos, S., and Basbaum, A. I., 1986, Benzidine dihydrochloride as a chromogen for single- and double-label light and electron microscopic immunocytochemical studies, J. Histochem. Cytochem. 34:1047–1056.PubMedCrossRefGoogle Scholar
  37. Larsson, L.-L., 1981, A novel immunocytochemical model system for specificity and sensitivity screening of anitsera against multiple antigens, J. Histochem. Cytochem. 29:408–410.PubMedCrossRefGoogle Scholar
  38. Larsson, L.-L., 1983, Methods for immunocytochemistry of neurohormonal peptides, in: Handbook of Chemical Neuroanatomy, Volume 1, Methods in Chemical Neuroanatomy (A. Bjorklund and T. Hokfelt, eds.), Elsevier, Amsterdam, pp. 147–209.Google Scholar
  39. Leranth, C., and Fehlér, E., 1983, Synaptology and sources of vasoactive intestinal polypeptide (VIP) and substance P (SP) containing axons of the cat celiac ganglion. (An experimental electron microscopic immunohistochemical study), Neuroscience 10:947–958.PubMedCrossRefGoogle Scholar
  40. Leranth, C., and Frotscher, M., 1986a, Synaptic connections of cholecystokinin-immunoreactive neurons and terminals in the rat fascia dentata: A combined light and electron microscopic study, J. Comp. Neurol. 254:51–64.PubMedCrossRefGoogle Scholar
  41. Leranth, C., and Frotscher, M., 1986b, GABAergic input of cholecystokinin-immunoreactive neurons in the hilar region of the rat hippocampus: An electron microscopic double im-munostaining study, Histochemistry 86:287–290.CrossRefGoogle Scholar
  42. Leranth, C., and Frotscher, M., 1987, Cholinergic innervation of hippocampal GAD- and so-matostatin-immunoreactive commissural neurons: Electron microscopic double immuno-staining combined with retrograde tracer technique, J. Comp. Neurol. 261:33–47.PubMedCrossRefGoogle Scholar
  43. Leranth, C., Williams, T. H., Chretien, M., and Palkovits, M., 1980a, Ultrastructural investigation of ACTH immunoreactivity in arcuate and supraoptic nuclei of the rat, Cell. Tissue Res. 210:11–19.PubMedCrossRefGoogle Scholar
  44. Leranth, C., Williams, T. H., Jew, J. Y., and Arimura, A., 1980b, Immuno-electron microscopic identification of somatostatin cells and axons of guinea pig sympathetic ganglia, Cell. Tissue Res. 212:83–89.PubMedCrossRefGoogle Scholar
  45. Leranth, C., Jew, J. Y., Williams, T. H., and Palkovits, M., 1981, Stria terminalis axons ending on substance P and neurotensin-containing cells of rat central amygdaloid nucleus: An electron microscopic immunocytochemical study, Neuropeptides 1:261–272.CrossRefGoogle Scholar
  46. Leranth, C., Sakamoto, H., MacLusky, N.J., Shanabrough, M., and Naftolin, F., 1985a, Application of avidin—ferritin and peroxidase as contrasting electron-dense markers for simultaneous electron microscopic immunocytochemical labelling of glutamic acid decarboxylase and tyrosine hydroxylase in the rat arcuate nucleus, J. Histochem. 82:165–168.CrossRefGoogle Scholar
  47. Leranth, C., MacLusky, N. J., Sakamoto, H., Shanabrough, M., and Naftolin, F., 1985b, Glutamic acid decarboxylase-containing axons synapse on LHRH neurons in the rat medial preoptic area, Neuroendocrinology 40:536–539.PubMedCrossRefGoogle Scholar
  48. Leranth, C., MacLusky, N., and Naftolin, F., 1985c, Synaptic inter-connections between LHRH, GAD, TH and ACTH hypothalamic neurons involved in the control of gonadotrophin release in the rat, Neurosci. Lett. Suppl. 22:598.Google Scholar
  49. Leranth, C., MacLusky, N. J., and Naftolin, F., 1986, Inter-connections between neurotransmitter and neuropeptide containing neurons involved in gonadotrophin release in rat, in: Neural and Endocrine Peptides and Receptors (T. W. Moody, ed.), Plenum Press, New York, pp: 177–193.CrossRefGoogle Scholar
  50. Leranth, C., MacLusky, N.J., Shanabrough, M., and Naftolin, F., 1988a, Immunohistochemical evidence for synaptic connections between proopiomelanicortin immunoreactive axons and LHRH neurons in the preoptic area of the rat, Brain Res. 449:167–176.PubMedCrossRefGoogle Scholar
  51. Leranth, C., MacLusky, N. J., Sharabrough, M., and Naftolin, F., 1988b, Catecholaminergic innervation of LHRH and GAD immunoreactive neurons in the rat medial preoptic area: an electron microscopic double immunostaining and degeneration study, Neuroendocrinology 48:591–602.PubMedCrossRefGoogle Scholar
  52. Levey, A. I., Bolam, J. P., Rye, D. B., Hallanger, A. E., Demuth, R. M., Mesulam, M. M., and Wainer, B. H., 1986, A light and electron microscopic procedure for sequential double antigen localization using diaminobenzidine and benzidine dihydrochloride, J. Histochem. Cytochem. 34:1449–1457.PubMedCrossRefGoogle Scholar
  53. Liposits, Z., Görcs, T., Török, A., Domány, S., and Sétáló, G., 1983, Simultaneous localization of two different tissue antigens based on the silver intensified PAP-DAB and the traditional PAP-DAB methods, Acta Morphol. Acad. Sci. Hung. 31:356–369.Google Scholar
  54. Liposits, Z., Sétáló, G., and Flerkó, B., 1984, Application of the silver-gold intensified 3,3’-diaminobenzidine chromogen to the light and electron microscopic detection of the LHRH system of the rat brain, Neuroscience 13:513–525.PubMedCrossRefGoogle Scholar
  55. Liposits, Z., Phelix, C., and Pauli, W. K., 1986a, Adrenergic innervation of corticotropin releasing factor (CRF)-synthesizing neurons in the hypothalamic paraventricular nucleus of the rat. A combined light and electron microscopic immunocytochemical study, Histochemistry 84:201–205.PubMedCrossRefGoogle Scholar
  56. Liposits, Z., Sherman, D., Phelix, C., and Pauli, W. K., 1986b, A combined light and electron microscopic immunocytochemical method for the simultaneous localization of multiple tissue antigens: TH immunoreactive innervation of CRF synthesizing neurons in the paraventricular nucleus of the rat, Histochemistry 85:95–106.PubMedCrossRefGoogle Scholar
  57. MacMillan, F. M., and Cuello, A. C., 1986, Monoclonal antibodies in neurohistochemistry: The state of the art, in: Neurohistochemistry: Modern Methods and Applications (P. Panula, H. Pai-varinta, and S. Soinila, eds.), Alan R. Liss, New York, pp. 49–74.Google Scholar
  58. McLean, D. C., and Singer, S. J., 1970, A general method for the specific staining of intracellular antigens with ferritin—antibody conjugates, Proc. Natl. Acad. Sci. U.S.A. 65:122–128.PubMedCrossRefGoogle Scholar
  59. McLean S., Skirboll, L. R., and Pert, C. B., 1983, Opiatergic projection from the bed nucleus to the habenula: Demonstration by a novel radioimmunohistochemical method, Brain Res. 278:255–257.PubMedCrossRefGoogle Scholar
  60. McLean, S., Skirboll, L. R., and Pert, C. B., 1985, Comparison of substance P and enkephalin distribution in rat brain: An overview using radioimmunocytochemistry, Neuroscience 14:837–852.PubMedCrossRefGoogle Scholar
  61. Mesulam, M.-M., and Rosene, D. L., 1979, Sensitivity in horseradish peroxidase neurohistochemistry: A comparative and quantitative analysis of nine methods, J. Histochem. Cytochem. 27:763–773.PubMedCrossRefGoogle Scholar
  62. Milner, T. A., and Pickel, V. M., 1986, Neurotensin in the rat parabrachial region: Ultrastructural localization and extrinsic sources of immunoreactivity, J. Comp. Neurol. 247:326–343.PubMedCrossRefGoogle Scholar
  63. Milner, T. A., Pickel, V. M., Chan, J., Massari, V. J., Oertel, W. H., Park, D. H., Joh, T. H., and Reis, D. J., 1987, Adrenaline neurons in the rostral ventrolateral medulla: II. Synaptic relationships with GABAergic terminals, Brain Res. 411:46–57.PubMedCrossRefGoogle Scholar
  64. Molin, S.-O., Nygren, H., and Dolonius, L., 1978, A new method for the study of glutaralde-hyde-induced cross-linking properties in proteins with special reference to the reaction with amino groups, J. Histochem. Cytochem. 26:412–414.PubMedCrossRefGoogle Scholar
  65. Painter, R. G., Tokuyasu, K. T., and Singer, S. J., 1973, Immunoferritin localization of intracellular antigens: The use of ultracryotomy to obtain ultrathin sections suitable for direct immunoferritin staining, Proc. Natl. Acad. Sci. U.S.A. 70:1649–1653.PubMedCrossRefGoogle Scholar
  66. Pearson, A. A., and O’Neil, S. L., 1958, A silver gelatine method for staining nerve fibers, Anat. Rec. 95:297–301.CrossRefGoogle Scholar
  67. Petrusz, P., 1983, Essential requirements for the validity of immunocytochemical staining procedures, J. Histochem. Cytochem. 34:177–179.CrossRefGoogle Scholar
  68. Phil, E., 1967, Ultrastructural localization of heavy metals by a modified sulfide silver method, Histochemie 10:126–139.CrossRefGoogle Scholar
  69. Pickel, V. M., 1981, Immunocytochemical methods, in: Neuroanatomical Tract-Tracing Methods (L. Heimer and M. J. RoBards, eds.), Plenum Press, New York, pp. 483–509.CrossRefGoogle Scholar
  70. Pickel, V. M., 1985, Ultrastructure of central catecholaminergic neurons, in: Neurohistochemistry Today (P. Peuula, H. Paivarimpa, and S. Soiuila, eds.), Alan R. Liss, New York, pp. 379–424.Google Scholar
  71. Pickel, V. M., and Beaudet, A., 1984, Combined use of autoradiography and immunocytochemical methods to show synaptic interactions between chemically defined neurons, in: Immu-nolabeling for Electron Microscopy (J. M. Polak and J. M. Varnell, eds.), Elsevier, Amsterdam, pp. 259–265.Google Scholar
  72. Pickel, V. M., and Teitelman, G., 1983, Light and electron microscopic localization of single and multiple antigens, in: Histochemical and Ultrastructural Identification of Monoamine Neurons (J. Furness and M. Costa, eds.), John Wiley & Sons, New York, pp. 79–109.Google Scholar
  73. Pickel, V., Chan, J., Joh, T., and Massari, V., 1984, Catecholaminergic neurons in the medial nuclei of the solitary tracts receive direct synapses from GABA-gabaergic terminals: Combined colloidal gold and peroxidase labeling of synthesizing enzymes, Soc. Neurosci. Abstr. 1:537.Google Scholar
  74. Pickel, V. M., Chan, J., and Milner, T. A., 1986a, Autoradiographic detection of (125-I)-second-ary antiserum: A sensitive light and electron microscopic labeling method compatible with peroxidase immunocytochemistry for dual localization of neuronal antigens, J. Histochem. Cytochem. 34:707–718.PubMedCrossRefGoogle Scholar
  75. Pickel, V. M., Chan, J., and Ganten, D., 1986b, Dual peroxidase and colloidal gold-labeling study of angiotensin converting enzyme and angiotensin-like immunoreactivity in the rat subfornical organ, J. Neurosci. 6:2457–2469.PubMedGoogle Scholar
  76. Pickel, V. M., Chan, J., Park, D. H., Joh, T. H., and Milner, T. A., 1986c, Ultrastructural localization of phenylethanolamine N-methyltransferase in sensory and motor nuclei of the vagus nerve, J. Neurosci. Res. 15:439–455.PubMedCrossRefGoogle Scholar
  77. Pickel, V. M., Joh, T. H., and Chan, J., 1988a, Dual ultrastructural localization of choline acetyl-transferase and tyrosine hydroxylase in the rat caudate nucleus, Brain Res. (in preparation).Google Scholar
  78. Pickel, V. M., Chan, J., and Milner, T. A., 1988b, Cellular substrates for interactions between neurons containing PN MT and GABA in the nuclei of solitary tracts, J. Comp. Neurol, (in press).Google Scholar
  79. Polak, J. M., and Van Noorden, S. (eds.), 1983, Immunocytochemistry: Practical Applications in Pathology and Biology, Wright, Bristol.Google Scholar
  80. Priestley, C. V., and Cuello, A. C., 1982, Co-existence of neuroactive substances as revealed by immunohistochemistry with monoclonal antibodies, in: Cotransmission (A. C. Cuello, ed.), Macmillan, London, pp. 117–128.Google Scholar
  81. Roth, J., 1982, The preparation of protein A-gold complexes with 3 nm and 15 nm gold particles and their use in labeling multiple antigens on ultrathin sections, Histochem J. 14:791–801.PubMedCrossRefGoogle Scholar
  82. Roth, J., Bendayan, M., and Orei, L., 1978, Ultrastructural localization of intracellular antigens by the use of protein A-gold complex, J. Histochem. Cytochem. 26:1074–1081.PubMedCrossRefGoogle Scholar
  83. Salpeter, M. M., Bachman, L., and Salpeter, E. E., 1969, Resolution in electron microscopic autoradiography, J. Cell Biol. 41:1–20.PubMedCrossRefGoogle Scholar
  84. Salpeter, M. M., Fertuck, H. C., and Salpeter, E. E., 1977, Resolution in electron microscopic autoradiography III. Iodine-125, the effect of heavy metal staining and reassessment of critical parameters, J. Cell. Biol. 72:161–173.PubMedCrossRefGoogle Scholar
  85. Schachner, M., Hedley-Whyte, E. T., Hse, D. W., Schoonmaker, G., and Bignami, A., 1977, Ultrastructural localization of glial fibrillary acidic protein in mouse cerebellum by immu-noperoxidase labeling, J. Cell Biol. 75:67–73.PubMedCrossRefGoogle Scholar
  86. Singer, R. S., and Schick, A. I., 1961, The properties of specific stains for electron microscopy prepared by conjugation of antibody with ferritin, J. Biophys. Biochem. Cytol. 9:519–537.PubMedCrossRefGoogle Scholar
  87. Slot, J., and Gueze, H., 1981, Sizing of protein A-colloidal gold probes for immuno-electron microscopy, J. Cell. Biol. 90:533–536.PubMedCrossRefGoogle Scholar
  88. Somogyi, P., and Takagi, H., 1982, A note on the use of picric acid paraformaldehyde—glutar-aldehyde fixative for correlated light and electron microscopic immunocytochemistry, Neuroscience 7:1779–1784.PubMedCrossRefGoogle Scholar
  89. Stathis, E. C., and Fabrikanos, A., 1958, Preparation of colloidal gold, Chem. Ind. 27:860–861.Google Scholar
  90. Steinbusch, H. W. M., Verhofstaad, A. A. J., and Joosten, H. W. J., 1978, Localization of serotonin in the central nervous system by immunohistochemistry: Description of a specific and sensitive technique and some applications. Neuroscience 3:811–819.PubMedCrossRefGoogle Scholar
  91. Sternberger, L. A., 1979, Immunocytochemistry, John Wiley & Sons, New York, Chicago, Brisbane, Toronto.Google Scholar
  92. Sternberger, L. A., and Joseph, S. H., 1979, The unlabeled antibody method. Contrasting color staining of paired pituitary hormones without antibody removal, J. Histochem. Cytochem. 27:1424–1429.PubMedCrossRefGoogle Scholar
  93. Sternberger, L. A., Hardy, P. H., Cuculis, J. J., and Meyers, H. G., 1970, The unlabeled antibody enzyme method of immunohistochemistry preparation and properties of soluble antigen—antibody complex (Horseradish peroxidase—antiperoxidase) and its use in identification of spirochetes. J. Histochem. Cytochem. 18:315–333.PubMedCrossRefGoogle Scholar
  94. Tapia, F., Varndell, I., Robert, M., DeMey, J., and Polak, J., 1983, Double immunogold staining method for simultaneous ultrastructural localization of regulatory peptides, J. Histochem. Cytochem. 31:977–981.PubMedCrossRefGoogle Scholar
  95. Tryer, N. M., and Bell, E. M., 1974, The intensification of cobalt-filled neuron profiles using a modification of Timm’s sulphide silver method, Brain Res. 73:151–155.CrossRefGoogle Scholar
  96. van den Pol, A. N., 1984, Colloidal gold and biotin-avidin conjugates as ultrastructural markers for neural antigens, Q.J. Exp. Physiol. 69:1–33.PubMedGoogle Scholar
  97. van den Pol, A. N., 1985a, Silver-intensified gold and peroxidase as dual ultrastructural im-munolabels for pre- and postsynaptic neurotransmitters, Science 228:332–335.PubMedCrossRefGoogle Scholar
  98. van den Pol, A. N., 1985b, Dual ultrastructural localization of two neurotransmitter-related antigens: Colloidal gold-label neurophysin-immunoreactive supraoptic neurons receive peroxidase-labeled glutamate decarboxylase- or gold-labeled GABA-immunoreactive synapses, J. Neurosci. 11:2940–2954.Google Scholar
  99. van den Pol, A. N., and Görcs, T., 1985, Suprachiasmatic nucleus, synaptic relationships: a dual ultrastructural immunocytochemistry study with peroxidase and gold substituted silver peroxidase, Soc. Neurosci. Abstr. 11:34.Google Scholar
  100. van den Pol, A. N., and Görcs, T., 1986, Synaptic relationships between neurons containing vasopressin, gastrin releasing hormone, VIP, and GAD immunoreactivity in the rat suprachiasmatic nucleus: Dual ultrastructural immunocytochemistry with gold substituted silver peroxidase, J. Comp. Neurol. 252:507–521.PubMedCrossRefGoogle Scholar
  101. van den Pol, A. N., Smith, A. D., and Powell, J. F., 1985, GABA axons in synaptic contact with dopamine neurons in the substantia nigra: Double immunocytochemistry with biotin—peroxidase and protein A—colloidal gold, Brain Res. 348:146–154.PubMedCrossRefGoogle Scholar
  102. Verhofstaad, A. A. J., Steinbusch, H. W. M., Joosten, H. W. J., Penke, B., Varga, J., and Goldstein, M., 1983, Immunocytochemical localization of noradrenaline, adrenaline and serotonin, in: Immunocytochemistry. Practical Applications in Pathology and Biology. J. M. Polak and S. Van Noorden, eds.), Wrigle, Bristol, pp. 143–167.Google Scholar
  103. Williams, T. H., and Jew, J. Y., 1975, An improved method for perfusion fixation of neural tissues for electron microscopy, Tissue Cell 7:407–418.PubMedCrossRefGoogle Scholar
  104. Zaborszky, L., Heimer, L., Eckenstein, F., and Leranth, C., 1986, GABAergic input to cholinergic forebrain neurons: An ultrastructural study using retrograde tracing of HRP and double immunolabeling, J. Comp. Neurol. 250:282–295.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1989

Authors and Affiliations

  • Csaba Leranth
    • 1
  • Virginia M. Pickel
    • 2
  1. 1.Section of Neuroanatomy and Department of Obstetrics and GynecologyYale University School of MedicineConnecticutUSA
  2. 2.Department of Neurology and Neuroscience, Laboratory of NeurobiologyCornell University Medical CollegeNew YorkUSA

Personalised recommendations