Immunocytochemical Labeling Methods and Related Techniques for Ultrastructural Analysis of Neuronal Connectivity

  • Patrizia Aimar
  • Laura Lossi
  • Adalberto Merighi


We describe a series of techniques based on the use of immunocytochemical labeling methods for the study of the neurochemistry and connectivity of cells within the central nervous system (CNS). According to our current view, neurons communicate to each other mainly at the level of synapses. Although it is now widely accepted that nonsynaptic neuron-to-neuron communication and neuron-to-glia crosstalk also occur, ultrastructural localization of transmitters–modulators at synapses and analysis of neuronal connections still remain major challenges for a correct understanding of the way in which neuronal networks are organized and operate.


Immunogold Label Superficial Dorsal Horn Neuronal Connectivity Gold Label Glial Fibrillary Acidic Protein Immunoreactivity 
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  1. 1.
    Aimar, P., I. Barajon, and A. Merighi. 1998. Ultrastructural features and synaptic connections of NADPH-diaphorase positive neurones in the rat spinal cord. Eur. J. Anat. 2:27–34.Google Scholar
  2. 2.
    Aimar, P., L. Lossi, and A. Merighi. 1997. Immunogold labeling for transmission electron microscopy: exploring new frontiers. Cell Vision 4:394–407.Google Scholar
  3. 3.
    Aimar, P., L. Pasti, G. Carmignoto, and A. Merighi. 1998. Nitric oxide-producing islet cells modulate the release of sensory neuropeptides in the rat substantia gelatinosa. J. Neurosci. 18:10375–10388.PubMedGoogle Scholar
  4. 4.
    Alvarez, F.J., A.M. Kavookjian, and R. Light. 1992. Synaptic interactions between GABA-immunoreactive profiles and the terminals of functionally defined myelinated nociceptors in the monkey and cat spinal cord. J. Neurosci. 12:2901–2917.PubMedGoogle Scholar
  5. 5.
    Basbaum, A.I. 1989. A rapid and simple silver enhancement procedure for ultrastructural localization of the retrograde tracer WGAapoHRP-Au and its use in double-label studies with post-embedding immunocytochemistry. J. Histochem. Cytochem. 37:1811–1815.PubMedGoogle Scholar
  6. 6.
    Beier, K. and H.D. Fahimi. 1985. Automatic determination of labeling density in protein A-gold immunocytochemical preparations using an image analyzer. Application to peroxisomal enzymes. Histochemistry 82:99–100.PubMedCrossRefGoogle Scholar
  7. 7.
    Bendayan, M. 1984. Protein A-gold electron microscopic immunocytochemistry: methods, applications, and limitations. J. Electron Microsc. Tech. 1:243–270.CrossRefGoogle Scholar
  8. 8.
    Bendayan, M. 1987. Introduction of the protein G-gold complex for high-resolution immunocytochemistry. J. Electron Microsc. Tech. 6:7–13.CrossRefGoogle Scholar
  9. 9.
    Bendayan, M., A. Nanci, and F.W.K. Kan. 1987. Effect of tissue processing on colloidal gold cytochemistry. J. Histochem. Cytochem. 35:983–996.PubMedGoogle Scholar
  10. 10.
    Bendayan, M. and M. Zollinger. 1983. Ultrastructural localization of antigenic sites on osmium-fixed tissues appliying the protein A-gold technique. J. Histochem. Cytochem. 31:101–109.PubMedGoogle Scholar
  11. 11.
    Blackstad, T.W., T. Karagulle, and O.P. Ottersen. 1990. Morforel, a computer program for two-dimensional analysis of micrographs of biological specimens, with emphasis on immunogold preparations. Comput. Biol.Med. 20:15–34.PubMedCrossRefGoogle Scholar
  12. 12.
    Bolam, J.P., C.A. Ingham, and A.D. Smith. 1984. The section-Golgi-impregnation procedure-3. Combination of Golgi-impregnation with enzyme histochemistry and electron microscopy to characterize acetylcholinesterase-containing neurons in the rat neostriatum. Neuroscience 12:711–718.PubMedCrossRefGoogle Scholar
  13. 13.
    Bouwens, L. and G. Kloppel. 1994. Cytochemical localization of NADPH-diaphorase in the four types of pancreatic islet cell. Histochemistry 101:209–214.PubMedCrossRefGoogle Scholar
  14. 14.
    Broman, J. and F. Ådahl. 1994. Evidence for vesicular storage of glutamate in primary afferent terminals. Neuroreport 5:1801–1804.PubMedCrossRefGoogle Scholar
  15. 15.
    Broman, J., J. Westman, and O.P. Ottersen. 1990. Ascending afferents to lateral cervical nucleus are enriched in glutamate-like immunoreactivity: a combined anterograde transport-immunogold study in the cat. Brain Res. 520:178–191.PubMedCrossRefGoogle Scholar
  16. 17.
    Brorson, S.H. and F. Skjorten. 1996a. Improved tical nique for immunoelectron microscopy. How to prepare epoxy resin to obtain approximately the same immunogold labeling for epoxy sections as for acrylic sections without any etching. Micron 27:211–217.PubMedCrossRefGoogle Scholar
  17. 17.
    Brorson, S.H. and F. Skjorten. 1996b. The theoretical relationship of immunogold labeling on acrylic sections and epoxy sections. Micron 27:193–201.PubMedCrossRefGoogle Scholar
  18. 18.
    Causton, B.E. 1984. The choice of resins for electron immunocytochemistry, p. 29–36. In J.M. Polak and I.M. Varndell (Eds.), Immunolabelling for Electron Microscopy. Elsevier Publishing, Amsterdam.Google Scholar
  19. 19.
    Chang, J.P. and M. Yokama. 1970. A modified section freeze substitution technique. J. Histochem. Cytochem. 18:683–684.Google Scholar
  20. 20.
    Cheng, P.Y., L.Y. Liu Chen, C. Chen, and V.M. Pickel. 1996. Immunolabeling of Mu opioid receptors in the rat nucleus of the solitary tract: extrasynaptic plasmalemmal localization and association with Leu5-enkephalin. J. Comp. Neurol. 371:522–536.PubMedCrossRefGoogle Scholar
  21. 21.
    Cheng, P.Y., A. Moriwaki, J.B. Wang, G.R. Uhl, and V.M. Pickel. 1996. Ultrastructural localization of muopioid receptors in the superficial layers of the rat cervical spinal cord: extrasynaptic localization and proximity to Leu5-enkephalin. Brain Res. 731:141–154.PubMedCrossRefGoogle Scholar
  22. 22.
    Cruz, F., D. Lima, and A. Coimbra. 1987. Several morphological types of terminal arborizations of primary afferents in laminae I–II of the rat spinal cord, as shown after HRP labeling and Golgi impregnation. J. Comp. Neurol. 261:221–236.PubMedCrossRefGoogle Scholar
  23. 23.
    De Zeeuw, C.I., J.C. Holstege, F. Calkoen, T.J.H. Ruigrok, and J. Voogd. 1988. A new combination of WGA-HRP anterograde tracing and GABA immunocytochemistry applied to afferents of the cat inferior olive at the ultrastructural level. Brain Res. 447:369–375.PubMedCrossRefGoogle Scholar
  24. 24.
    Dudek, R.W. and A.F. Boyne. 1986. An excursion through the ultrastructural world of quick-fozen pancreatic islets. Am. J. Anat. 175:217–243.PubMedCrossRefGoogle Scholar
  25. 25.
    Dudek, R.W., A.F. Boyne, and M. Freinkel. 1981. Quick-freeze fixation and freeze-drying of isolated rat pancreatic islets: application to the ultrastructural localization of inorganic phosphate in the pancreatic beta cells. J. Histochem. Cytochem. 29:321–325.PubMedGoogle Scholar
  26. 26.
    Dudek, R.W., G.V. Childs, and A.F. Boyne. 1982. Quick-freezefixation and freeze drying for electron for high quality morphology and immunocytochemistry at the ultrastructural level: application to pancreatic beta cells. J. Histochem. Cytochem. 30:129–138.PubMedGoogle Scholar
  27. 27.
    Dudek, R.W., I.M. Varndell, and J.M. Polak. 1984. Quick-freeze fixation and freeze drying for electron microscopic immunocytochemistry, p. 235–248. In J.M. Polak and I.M. Varndell (Eds.), Immunolabelling for Electron Microscopy. Elsevier Publishers, Amsterdam.Google Scholar
  28. 28.
    Eneström, S. and B. Kniola. 1990. Quantitiative ultrastructural immunocytochemistry using a computerized image analysis system. Stain Technol. 65:263–278.PubMedGoogle Scholar
  29. 29.
    Eneström, S. and B. Kniola. 1995. Resin embedding for quantitative immunoelectron microscopy. A comparative computerized image analysis. Biotech. Histochem. 70:135–146.PubMedCrossRefGoogle Scholar
  30. 30.
    Faulk, W.P. and G.M. Taylor. 1971. An immunocolloid method for the electron microscope. Immunochemistry 8:1081–1083.PubMedCrossRefGoogle Scholar
  31. 31.
    Gilerovitch, H.G., G.A. Bishop, J.S. King, and R.W. Burry. 1995. The use of electron microscopic immunocytochemistry with silver-enhanced 1.4-nm gold particles to localize GAD in the cerebellar nuclei. J. Histochem. Cytochem. 43:337–343.PubMedGoogle Scholar
  32. 32.
    Gingras, D. and M. Bendayan. 1994. Compartmentalization of secretory proteins in pancreatic zymogen granules as revealed by immunolabeling on cryo-fixed and molecular distillation processed tissue. Biol. Cell. 81:153–163.PubMedCrossRefGoogle Scholar
  33. 33.
    Gomez, S. and A. Boyde. 1994. Correlated alkaline phosphatase histochemistry and quantitative backscattered electron imaging in the study of rat incisor ameloblasts and enamel mineralization. Microsc. Res. Tech. 29:29–36.PubMedCrossRefGoogle Scholar
  34. 34.
    Gong, L.W., Y.-Q. Ding, D. Wang, H.X. Zheng, B.Z. Qin, J.S. Li, T. Kaneko, and N. Mizuno. 1997. GABAergic synapses on mu-opioid receptor-expressing neurons in the superficial dorsal horn: an eletron microscopy study in the cat spinal cord. Neurosci. Lett. 227:33–36.PubMedCrossRefGoogle Scholar
  35. 35.
    Gracy, K.N. and V.M. Pickel. 1996. Ultrastructural immunocytochemical localization of the N-methyl-D-aspartate receptor and tyrosine hydroxylase in the shell of the rat nucleus accumbens. Brain Res. 739:169–181.PubMedCrossRefGoogle Scholar
  36. 36.
    Gundersen, V., O. Shupliakov, L. Brodin, O.P. Ottersen, and J. Storm-Mathisen. 1995. Quantification of excitatory amino acid uptake at intact glutamatergic synapses by immunocytochemistry of exogenous D-aspartate. J. Neurosci. 15:4417–4428.PubMedGoogle Scholar
  37. 37.
    Gustincich, S., A. Feigenspan, D.K. Wu, L.J. Koopman, and E. Raviola. 1997. Control of dopamine release in the retina: a transgenic approach to neural networks. Neuron 18:723–726.PubMedCrossRefGoogle Scholar
  38. 38.
    Hainfeld, J.F. and F.R. Furuya. 1992. A 1.4 nm gold gluster covalently attached to antibodies improves immunolabeling. J. Histochem. Cytochem. 40:177–184.PubMedGoogle Scholar
  39. 39.
    Hainfeld, J.F. and R.D. Powell. 1997. Nanogold technology: new frontiers in gold labeling. Cell Vision 4:408–432.Google Scholar
  40. 40.
    Hayat, M.A. 1977. Principles and techniques of electron microscopy. Vol. 1. Biological Applications. Edward Arnold, New York.Google Scholar
  41. 41.
    Holstege, J.C. and J.M. Vrensen. 1988. Anterograde tracing in the brain using autoradiography and HRP-histochemistry. A comparison at the ultrastructural level. J. Microsc. 150:233–243.PubMedGoogle Scholar
  42. 42.
    Howell, K.E., U. Reuter-Carlson, E. Devaney, J.P. Luzio, and S.D. Fuller. 1987. One antigen one gold? A quantitative analysis of immunogold labelling of plasma membrane 5’-nucleotidase in frozen thin sections. Eur. J. Cell Biol. 44:318–326.PubMedGoogle Scholar
  43. 43.
    Hsu, S.M., L. Raine, and H. Fanger. 1981. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques. A comparison between ABC and unlabeled antibody (PAP) procedures. J. Histochem. Cytochem. 29:577–587.PubMedGoogle Scholar
  44. 44.
    Huiz, J.M., M.L. Campos, R.H. Helfert, and R.A. Altschuler. 1996. Silver intensification of immunocolloidal gold on ultrathin plastic sections applied to the study of the neuronal distribution of GABA and glycine. J. Hirnforsch. 37:51–56.Google Scholar
  45. 45.
    Kechagias, S. and J. Broman. 1994. Compartmentation of glutamate and glutamine in the lateral cervical nucleus: further evidence for glutamate as a spinocervical tract neurotransmitter. J. Comp. Neurol. 340:531–540.PubMedCrossRefGoogle Scholar
  46. 46.
    Keller, G.A., K.T. Tokuyasu, A.H. Dutton, and S.J. Singer. 1984. An improved procedure for immunoelectronmicroscopy: ultrathin plastic embedding of immunolabelled ultrathin frozen sections. Proc. Natl. Acad. Sci. USA 81:5744–5747.PubMedCrossRefGoogle Scholar
  47. 47.
    Knyihar-Csillik, E., B. Csillik, and A.B. Oestreicher. 1992. Light and electron microscopic localization of B-50 (GAP43) in the rat spinal cord during transganglionic degenerative atrophy and regeneration. J. Neurosci. Lett. 32:93–375.Google Scholar
  48. 48.
    Knyihar-Csillik, E., B. Csillik, and P. Rakic. 1982. Ultrastructure of normal and degenerating glomerular terminals of dorsal root axons in the substantia gelatinosa of the Rhesus monkey. J. Comp. Neurol. 210:357–375.PubMedCrossRefGoogle Scholar
  49. 49.
    Krenacs, T. and L. Dux. 1994. Silver-enhanced immunogold labeling of calcium-ATPase in sarcoplasmic reticulum of skeletal muscle [letter, comment]. J. Histochem. Cytochem. 42:967–968.PubMedGoogle Scholar
  50. 50.
    Krenacs, T., M. van Dartel, E. Lindhout, and M. Rosendaal. 1997. Direct cell/cell communication in the lymphoid germinal center: connexin43 gap junctions functionally couple follicular dendritic cells to each other and to B lymphocytes. Eur. J. Immunol. 27:1489–1497.PubMedCrossRefGoogle Scholar
  51. 51.
    Light, A.R. and A.M. Kavookjian. 1988. Electron microscopic localization of peptide-like immunoreactivity in labelled dorsal root terminals in the spinal substantia gelatinosa of the monkey, p. 57–69. In F. Cervero, G.J. Bennett, and P.M. Headley (Eds.), Processing of Sensory Information in the Superficial Dorsal Horn of the Spinal Cord. Plenum Press, New York.Google Scholar
  52. 52.
    Liou, W., H.J. Geuze, and J.W. Slot. 1996. Improving structural integrity of cryosections for immunogold labeling. Histochem. Cell Biol. 106:41–58.PubMedGoogle Scholar
  53. 53.
    Lue, J.H., Y.F. Jiang Shieh, J.Y. Shieh, and C.Y. Wen. 1996. The synaptic interrelationships between primary afferent terminals, cuneothalamic relay neurons and GABA-immunoreactive boutons in the rat cuneate nucleus. Neurosci. Res. 24:363–371.PubMedCrossRefGoogle Scholar
  54. 54.
    Maxwell, D.J., W.H. Christie, A.D. Short, J. Störm-Mathisen, and O.P. Ottersen. 1990. Central boutons of glomeruli in the spinal cord of the cat are enriched with L-glutamate-like immunoreactivity. Neuroscience 36:83–104.PubMedCrossRefGoogle Scholar
  55. 55.
    Maxwell, D.J., W.M. Christie, O.P. Ottersen, and J. Störm-Mathisen. 1990. Terminals of group Ia primary afferent fibers in Clarke’s column are enriched with L-glutamate-like immunoreactivity. Brain Res. 510:346–350.PubMedCrossRefGoogle Scholar
  56. 56.
    Maxwell, D.J., W.M. Christie, A.D. Short, and A.G. Brown. 1990. Direct observations of synapses between GABA-immunoreactive boutons and muscle afferent terminals in lamina VI of cat spinal cord. Brain Res. 530:215–222.PubMedCrossRefGoogle Scholar
  57. 57.
    Maxwell, D.J., R. Kerr, E. Jankowska, and J.S. Riddell. 1997. Synaptic connections of dorsal horn group II spinal interneurons: synapses formed with the interneurons and by their axon collaterals. J. Comp. Neurol. 380:51–69.PubMedCrossRefGoogle Scholar
  58. 58.
    Maxwell, D.J., O.P. Ottersen, and J. Storm-Mathisen.1995. Synaptic organization of excitatory and inhibitory boutons associated with spinal neurons which project through the dorsal columns of the cat. Brain Res. 676:103–112.PubMedCrossRefGoogle Scholar
  59. 59.
    Menco, B.P., A.M. Cunningham, P. Qasba, N. Levy, and R.R. Reed. 1997. Putative odour receptors localize in cilia of olfactory receptor cells in rat and mouse: a freeze-substitution ultrastructural study. J. Neurocytol. 26:297–312.PubMedCrossRefGoogle Scholar
  60. 60.
    Merighi, A. 1992. Post-embedding electron microscopic immunocytochemistry, p. 51–87. In J.M. Polak and J.V. Priestly (Eds.), Electron Microscopic Immunocytochemistry. Oxford University Press, London.Google Scholar
  61. 61.
    Merighi, A., F. Cruz, and A. Coimbra. 1992. Immunocytochemical staining of neuropeptides in terminal arborization of primary afferent fibers anterogradely labeled and identified at light and electron microscopic levels. J. Neurosci. Methods 42:105–113.PubMedCrossRefGoogle Scholar
  62. 62.
    Merighi, A. and J.M. Polak. 1993. Post-embedding immunogold staining, p. 229–264. In A.C. Cuello (Ed.), Immunohistochemistry II. John Wiley & Sons, London.Google Scholar
  63. 63.
    Merighi, A., J.M. Polak, and D.T. Theodosis. 1991. Ultrastructural visualization of glutamate and aspartate immunoreactivities in the rat dorsal horn with special reference to the co-localization of glutamate, substance P and calcitonin gene-related peptide. Neuroscience 40:67–80.PubMedCrossRefGoogle Scholar
  64. 64.
    Merighi, A., E. Raviola, and R.E Dacheux. 1996. Connections of two types of flat cone bipolars in the rabbit retina. J. Comp. Neurol. 371:164–178.PubMedCrossRefGoogle Scholar
  65. 65.
    Morris, J.E., D.V. Pow, and F.D. Shaw. 1989. Strategies for the ultrastructural study of peptide containing neurons, p. 83–124. In G. Fink and A.J. Harmar (Eds.), Neuropeptides: A Methodology. John Wiley & Sons, Chichester.Google Scholar
  66. 66.
    Negyessy, L., J. Takacs, I. Divac, and J. Hamori. 1994. A combined Golgi and post-embedding GABA and glutamate electron microscopic study of the nucleus dorsomedialis thalami of the rat. Neurobiology 2:83–104.Google Scholar
  67. 67.
    Nirenberg, M.J., J. Chan, R.A. Vaughan, G.R. Uhl, M.J. Kuhar, and V.M. Pickel. 1997. Immunogold localization of the dopamine transporter: an ultrastructural study of the rat ventral tegmental area. J. Neurosci. 17:5255–5262.PubMedGoogle Scholar
  68. 68.
    Ottersen, O.P. and C.R. Bramham. 1988. Quantitative electron microscopic immunocytochemistry of excitatory amino acids, p. 93–100. In E.A. Cavalheiro, J. Lehmann and L. Turski (Eds.), Frontiers in Excitatory Amino Acids Research. Alan R. Liss, New York.Google Scholar
  69. 69.
    Pease, D.C. 1964. Histological Techniques for Electron Microscopy. Academic Press, New York.Google Scholar
  70. 70.
    Phend, K.D., A. Rustioni, and R.J. Weinberg. 1995. An osmium-free method of epon embedment that preserves both ultrastructure and antigenicity for postembedding immunocytochemistry. J. Histochem. Cytochem. 43:283–292.PubMedGoogle Scholar
  71. 71.
    Pickel, V.M., J. Chan, and C. Aoki. 1993. Electron microscopic immunocytochemical labelling of endogenous and/or transported antigens in rat brain using silver intensified one-nanometre colloidal gold, p. 265–280. In A.C. Cuello (Ed.), Immunohistochemistry II. John Wiley & Sons, Chichester.Google Scholar
  72. 72.
    Posthuma, G., J.W. Slot, and H.J. Geuze. 1984. Immunocytochemical assays of amylase and chymotrypsinogen in rat pancreas secretory granules. Efficacy of using immunogold-labeled ultrathin cryosections to estimate relative protein concentrations. J. Histochem. Cytochem. 32:1028–1034.PubMedGoogle Scholar
  73. 73.
    Posthuma, G., J.W. Slot, and H.J. Geuze. 1987. Usefulness of the immunogold technique in quantitation of a soluble protein in ultra-thin sections. J. Histochem. Cytochem. 35:405–411.PubMedGoogle Scholar
  74. 74.
    Priestley, J.V. 1984. Pre-embedding ultrastructural immunocytochemistry: immunoenzyme techniques, p. 37–52. In J.M. Polak and I.M. Varndell (Eds.), Immunolabelling for Electron Microscopy. Elsevier Publishers, Amsterdam.Google Scholar
  75. 75.
    Priestley, J.V. and A.C. Cuello. 1989. Ultrastructural and neurochemical analysis of synaptic input to trigemino-thalamic projection neurones in lamina I of the rat: a combined immunocytochemical and retrograde labelling study. J. Comp. Neurol. 285:467–486.PubMedCrossRefGoogle Scholar
  76. 76.
    Pulczynski, S. and O. Myhre Jensen. 1994. Quantitation of immunogold with an interactive image analysis system: a new, practical approach to antibody-induced modulation, internalization and intracellular transport of surface antigens in viable hematopoietic cells. Anal. Quant. Cytol. Histol. 16:393–399.PubMedGoogle Scholar
  77. 77.
    Rèthelyi, M., A.R. Light, and E.R. Perl. 1989. Synaptic ultrastructure of functionally and morphologically characterized neurons of the superficial spinal dorsal horn of cat. J. Neurosci. 9:1846–1863.PubMedGoogle Scholar
  78. 78.
    Ribeiro-Da-Silva, A., J.V. Priestley, and A.C. Cuello. 1993. Pre-embedding ultrastructural immunocytochemistry, p. 181–228. In A.C. Cuello (Ed.), Immunohistochemistry II. John Wiley & Sons, Chichester.Google Scholar
  79. 79.
    Robards, A.W. and U.B. Sleytr. 1985. Low temperature methods in biological electron microscopy, p. 1–551. In A.M. Glauert (Ed.), Practical Methods in Electron Microscopy. Elsevier Publishers, Amsterdam.Google Scholar
  80. 80.
    Roth, J. 1984. The protein A-gold (pAg) technique—a qualitative and quantitative approach for antigen localization on thin sections, p. 107–134. In G.R. Bullock and P. Petrusz (Eds.), Techniques in Immunohistochemistry (Vol. I). Academic Press, London.Google Scholar
  81. 81.
    Roth, J. 1984. The protein A-gold technique for antigen localization in tissue sections by light and electron microscopy, p. 113–122. In J.M. Polak and I.M. Varndell (Eds.), Immunolabelling for Electron Microscopy. Elsevier Publishers, Amsterdam.Google Scholar
  82. 82.
    Roth, J. 1996. The silver anniversary of gold: 25 years of the colloidal gold marker system for immunocytochemistry and histochemistry. Histochem. Cell. Biol. 106:1–8.PubMedGoogle Scholar
  83. 83.
    Saha, S., T.E. Batten, and P.N. McWilliam. 1995. Glutamate-immunoreactivity in identified vagal afferent terminals of the cat: a study combining horseradish peroxidase tracing and post-embedding electron microscopic immunogold staining. Exp. Physiol. 80:193–202.PubMedGoogle Scholar
  84. 84.
    Schwarz, C. and Y. Schmitz. 1997. Projection from the cerebellar lateral nucleus to precerebellar nuclei in the mossy fiber pathway is glutamatergic: a study combining anterograde tracing with immunogold labeling in the rat. J. Comp. Neurol. 381:320–334.PubMedCrossRefGoogle Scholar
  85. 85.
    Sekerkova, G., Z. Katarova, F. Joo, J.R. Wolff, S. Prodan, and G. Szabo. 1997. Visualization of beta-galactosidase by enzyme and immunohistochemistry in the olfactory bulb of transgenic mice carrying the LacZ transgene. J. Histochem. Cytochem. 45:1147–1155.PubMedGoogle Scholar
  86. 86.
    Slot, J.W., G. Posthuma, L.Y. Chang, J.D. Crapo, and H.J. Geuze. 1989. Quantitative aspects of immunogold labeling in embedded and non-embedded sections. Am. J. Anat. 185:271–281.PubMedCrossRefGoogle Scholar
  87. 87.
    Somogyi, P. 1990. Synaptic connections of neurones identified by golgi impregnation: characterisation by immunocytochemical, enzyme histochemical and degeneration methods. J. Electron Microsc. Tech. 15:332–351.PubMedCrossRefGoogle Scholar
  88. 88.
    Somogyi, P., K. Halasy, J. Somogyi, J. Störm-Mathisen, and O.P. Ottersen. 1986. Quantification of immunogold labelling reveals enrichment of glutamate in mossy and parallel fibre terminals in cat cerebellum. Neuroscience 19:1045–1050.PubMedCrossRefGoogle Scholar
  89. 89.
    Song, Z.M., S.J. Brookes, and M. Costa. 1994. Characterization of alkaline phosphatase-reactive neurons in the guinea-pig small intestine. Neuroscience 63:1153–1167.PubMedCrossRefGoogle Scholar
  90. 90.
    Sternberger, L.A., P.J.J. Hardy, J.J. Cucculis, and H.G. Meyer. 1970. The unlabeled antibody-enzyme method of immunohisto-chemistry. Preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-anti-horseradish peroxidase) and its use in identification of spirochetes. J. Histochem. Cytochem. 18:315–333.PubMedGoogle Scholar
  91. 91.
    Stirling, J.W. 1990. Immuno-and affinity probes for electron microscopy: a review of labeling and preparation techniques. J. Histochem. Cytochem. 38:145–158.PubMedGoogle Scholar
  92. 92.
    Stirling, J.W. 1993. Use of tannic acid and silver enhancer to improve staining for electron microscopy and immunogold labeling. J. Histochem. Cytochem. 41:643–648.PubMedGoogle Scholar
  93. 93.
    Stirling, J.W. and P.S. Graff. 1995. Antigen unmasking for immunoelectron microscopy: labeling is improved by treating with sodium ethoxide or sodium metaperiodate, then heating on retrieval medium. J. Histochem. Cytochem. 43:115–123.PubMedGoogle Scholar
  94. 94.
    Tang, F.R., C.K. Tan, and E.A. Ling. 1995. The distribution of NADPH-d in the central grey region (lamina X) of rat upper thoracic spinal cord. J. Neurocytol. 24:735–743.PubMedCrossRefGoogle Scholar
  95. 95.
    Tokuyasu, K.T. 1984. Immuno-cryoultramicrotomy, p. 71–82. In J.M. Polak and I.M. Varndell (Eds.), Immunolabelling for Electron Microscopy. Elsevier Publishers, Amsterdam.Google Scholar
  96. 96.
    van Lookeren Campagne, M., A.B. Oestreicher, T.P. Van Der Krift, W.H. Gispen, and A.J. Verkleij. 1991. Freeze-substitution and lowicryl HM20 embedding of fixed rat brain: suitability for immunogold ultrastructural localization of neural antigens. J. Histochem. Cytochem. 39:1267–1279.PubMedGoogle Scholar

Copyright information

© Springer-Verlag New York, Inc. 2002

Authors and Affiliations

  • Patrizia Aimar
    • 1
  • Laura Lossi
    • 1
  • Adalberto Merighi
    • 1
    • 2
  1. 1.Department of Veterinary Morphophysiology-Neuroscience Research GroupUniversità degli Studi di TorinoTorinoItaly, EU
  2. 2.Rita Levi Montalcini Center for Brain RepairUniversità degli Studi di TorinoTorinoItaly, EU

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