Skip to main content
SpringerLink
Log in

Electron Microscopic Preembedding Double-Immunostaining Methods

  • Chapter
Neuroanatomical Tract-Tracing Methods 2

Abstract

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).

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 74.99
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  • 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.

    Article  CAS  Google Scholar 

  • 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.

    Article  Google Scholar 

  • Beaudet, A., 1982, High resolution radioautography of central 5-hydroxytryptamine (5-HT) neurons, J. Histochem Cytochem. 9:765–768.

    Article  Google Scholar 

  • Bendayan, M., 1982, Double immunocytochemical labeling applying to protein A-gold technique, J. Histochem. Cytochem. 30:81–85.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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 

  • Cuello, A. C. (ed.), 1983, IBRO Handbook Series: Methods in Neurosciences, Volume 3, Immunohis-tochemistry, John Wiley & Sons, Chichester.

    Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • DeMey, J., 1983, A critical review of light and electron microscopic immunocytochemical techniques used in neurobiology, J. Neurosci. Methods 7:1–18.

    Article  CAS  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • 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 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    CAS  Google Scholar 

  • 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.

    Article  Google Scholar 

  • Faulk, V. P., and Taylor, G. P., 1971, An immunocolloid method for electron microscope, Immunochemistry 8:1081–1083.

    Article  PubMed  CAS  Google Scholar 

  • Ferns, G., 1973, Controlled nucleation for the regulation of the particle size in nondisperse gold solutions, Nature 241:20–22.

    Google Scholar 

  • Fertuck, H. C., and Salpeter, M. M., 1974, Sensitivity in electron microscopic autoradiography for 125I, J. Histochem. Cytochem. 22:80–87.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Chapter  Google Scholar 

  • Gallyas, F., 1971, A principle for silver staining of tissue elements by physical development, Acta Morphol. Acad. Sci. Hung. 19:57–71.

    PubMed  CAS  Google Scholar 

  • Gallyas, F., 1982, Suppression of argyrophil III reaction by mercaptocompounds. Prerequisite for intensification of histochemical reactions by physical developers, Acta Histochem. 70:99–105.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • Haug, F. M., 1967, Electron microscopic localization of the zinc in hippocampal mossy fibre synapses by a modified sulfide procedure, Histochemie 8:355–368.

    Article  PubMed  CAS  Google Scholar 

  • Horisberger, M., 1982, Evaluation of colloidal gold as a cytochemical marker for transmission and scanning electron microscopy, Biol. Cell. 36:253–258.

    Google Scholar 

  • Horisberger, M., and Rosset, J., 1977, Colloidal gold, a useful marker for transmission and scanning electron microscopy, J. Histochem. Cytochem. 25:295–305.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  Google Scholar 

  • Hunt, S. P., and Mantyh, P. W., 1984, Radioimmunohistochemistry with 3H-biotin, Brain Res., 291:203–217.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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 

  • 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.

    Chapter  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • Pearson, A. A., and O’Neil, S. L., 1958, A silver gelatine method for staining nerve fibers, Anat. Rec. 95:297–301.

    Article  Google Scholar 

  • Petrusz, P., 1983, Essential requirements for the validity of immunocytochemical staining procedures, J. Histochem. Cytochem. 34:177–179.

    Article  Google Scholar 

  • Phil, E., 1967, Ultrastructural localization of heavy metals by a modified sulfide silver method, Histochemie 10:126–139.

    Article  Google Scholar 

  • 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.

    Chapter  Google Scholar 

  • 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 

  • 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 

  • 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 

  • 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 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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 

  • 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 

  • Polak, J. M., and Van Noorden, S. (eds.), 1983, Immunocytochemistry: Practical Applications in Pathology and Biology, Wright, Bristol.

    Google Scholar 

  • 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 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • Salpeter, M. M., Bachman, L., and Salpeter, E. E., 1969, Resolution in electron microscopic autoradiography, J. Cell Biol. 41:1–20.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • Slot, J., and Gueze, H., 1981, Sizing of protein A-colloidal gold probes for immuno-electron microscopy, J. Cell. Biol. 90:533–536.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • Stathis, E. C., and Fabrikanos, A., 1958, Preparation of colloidal gold, Chem. Ind. 27:860–861.

    Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • Sternberger, L. A., 1979, Immunocytochemistry, John Wiley & Sons, New York, Chicago, Brisbane, Toronto.

    Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  Google Scholar 

  • 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.

    PubMed  Google Scholar 

  • 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.

    Article  PubMed  Google Scholar 

  • 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 

  • 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 

  • 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.

    Article  PubMed  Google Scholar 

  • 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.

    Article  PubMed  Google Scholar 

  • 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 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Section of Neuroanatomy and Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Connecticut, 06510, USA

    Csaba Leranth

  2. Department of Neurology and Neuroscience, Laboratory of Neurobiology, Cornell University Medical College, New York, New York, 10021, USA

    Virginia M. Pickel

Authors
  1. Csaba Leranth
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Virginia M. Pickel
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. University of Virginia Medical Center, Charlottesville, Virginia, USA

    Lennart Heimer

Rights and permissions

Reprints and permissions

Copyright information

© 1989 Springer Science+Business Media New York

About this chapter

Cite this chapter

Leranth, C., Pickel, V.M. (1989). Electron Microscopic Preembedding Double-Immunostaining Methods. In: Heimer, L., Záborszky, L. (eds) Neuroanatomical Tract-Tracing Methods 2. Springer, Boston, MA. https://doi.org/10.1007/978-1-4757-2055-6_6

Download citation

  • DOI: https://doi.org/10.1007/978-1-4757-2055-6_6

  • Publisher Name: Springer, Boston, MA

  • Print ISBN: 978-1-4757-2057-0

  • Online ISBN: 978-1-4757-2055-6

  • eBook Packages: Springer Book Archive

Publish with us

Policies and ethics

Search

Navigation