Abstract
Few techniques have approached the high resolution afforded by immunoelectron microscopy using gold markers for the detection of specific cellular proteins and other molecules (1) or by post-embedding procedures (2). In principal, a primary antibody to a particular protein, which has been fixed, embedded, and placed on a grid, is identified by a host-specific secondary antibody conjugated to a gold particle of defined size. The gold particle identifying the targeted protein is then detected by electron microscope observation. This method has undergone a steady development over the past few years, because it uniquely meets the need to precisely assign macromolecules to specific locations and domains within both tissues and cells. It has also been used to reveal antigens that may be present in low or trace amounts and thus, has contributed to a greater understanding of functional specialization domains within cells and tissues. Its advantages over light microscopic immunocytochemistry and confocal immunofluorescence localization studies are that it can be carried out on very minute specimens and that it can provide a permanent record for quantitative analyses of multiple domains. Its disadvantage is that, because of the small tissue size, more sampling is needed and more expertise is required in handling, thus requiring more overall experimental time. Additionally, the operation of an electron microscope (EM) can result in prohibitive costs.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Verkleij A., and Leunissen, J. L. M. (1989) Immuno-Gold Labelling in Cell Biology. CRC Press, Boca Raton, FL.
Mayer, G., and Bendayan, M. (2001) Amplification methods for the imunolocalization of rare molecules in cells and tissues. Prog. Histochem. Cytochem. 36, 3–85.
Jensen, H. L., and Norrild, B. (1999) 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, 523–533.
Renno, W. M. (2001) Post-embedding double-gold labeling immunoelectron microscopic co-localization of neurotransmitters in the rat brain. Med. Sci. Monit. 7, 188–200.
Wang, X. S., Ong, W. Y., Lee, H. K., and Huganir, R. L. (2000) A light and electron microscopic study of glutamate receptors in the monkey subthalamic nucleus. J. Neurocytol. 29, 743–754.
Cahill, C. J., and Nayak, R. C. (2000) Immunoelectron microscopic detection of tissue ganglioside antigens. J. Immunol. Methods 238,45–53.
Farrell, C. L., and Pardridge, W. M. (1991) Ultrastructural localization of blood-brain barrier specific antibodies using immunogold-silver enhancement techniques. J. Neurosci. Methods 37, 103–110.
Cornford, E. M., and Hyman, S. (1999) Blood-brain barrier permeability to small and large molecules. Adv. Drug Delivery Rev. 36,145–163.
Leino, R. L., Gerhart, D. Z., and Drewes, L. R. (1999) Monocarboxylic acid transporter (MCT1) abundance in brains of suckling and adult rats: a quantitative electron microscopic immunogold study. Brain Res. Dev. Brain Res. 113,47–54.
Nico, B., Quondamatteo, F., Herken, R., et al. (1999) Developmental expression of ZO-1 antigen in the mouse blood-brain barrier. Brain Res. Dev. Brain Res. 114,161–169.
Gajkowska, B., and Mossakowski, M. J. (1997) Endothelin-loke immunoreactivitiy in hippocampus following transient global cerebral ischemia. II. The blood-brain interphase. Folia Neuropathol. 35, 49–59.
Easton, A. S., and Dorovini-Zis, K. (2001) The kinetics, function, and regulation of p-selectin expressed by human brain microvessel endothelial cells in primary culture. Microvasc. Res. 62, 335–345.
Sierralta, W. D. (2001) Immunoelectron microscopy in embryos. Methods 24, 61–69.
Paupard, M. C, Miller, A., Grant, B., Hirsh, D., and Hall, D. H. Immuno-EM localization of GFP-tagged yolk proteins in C. elegans using microwave fixation. J. Histochem Cytochem. 49, 949–956.
Rangell, L. K., and Keller, G. A. (2000) Application of microwave technology to the processing and immunolabeling of plastic-embedded and cryosections. J. Histochem. Cytochem. 48, 1153–1159.
Farrell, C. L., and Pardridge, W. M. (1991) Blood-brain barrier glucose transporter is asymmetrically distributed on brain capillary endothelial lumenal and ablumenal membranes: An electron microscopic immunogold study. Proc. Natl. Acad. Sei. USA 88, 5779–5783.
Ramandeep, Dikshit, K. L., and Raje, M. (2001) Optimization of immunogold labeling TEM. An ELISA-based method for rapid and convenient simulation of processing conditions for quantitative detection of antigen. J. Histochem. Cytochem. 49, 355–368.
Brorson, S. H. (1998) Comparison of the immunogold labeling of single light chains and whole immunoglobulins with anti-kappa on LR-white and epoxy sections. Micron 29,439–443.
Vorbrodt, A. W., Dobrogowska, D. H., Meeker, H. C, and Carp, R. I. (1999) Immunogold study of regional differences in the distribution of glucose transporter (GLUT-1) in mouse brain associated with physiological and accelerated aging and scrapie infection. J. Neurocytol. 28, 711–719.
Vorbrodt, A. W., Dobrogowska, D. H., Kozlowski, P., Tarnawski, M., Dumas, R., and Rabe, R. (2001) Effect of a single embryonic exposure to alcohol on glucose transporter (GLUT-1) distribution in brain vessels of aged mouse. J. Neurocytol. 30,167–174.
Vorbrodt, A. W., Dobrogowska, D. H., Tarnawski, M., Meeker, H. C, and Carp, R. I. (2001b) Quantitative immunogold study of glucose transporter (GLUT-1) in five brain regions of scrapie-infected mice showing obesity and reduced glucose tolerance. Acta Neuropathol. (Berl.) 102, 278–284.
Thorpe, J. R. (1999) The application of LR gold resin for immunogold labelling. Methods Mol. Biol. 117, 99–110.
Reynolds, E. S. (1963) The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17, 208–213.
Cornford, E. M., Hyman, S., Cornford, M. E., Landaw, E. M., and Delgado-Escueta, A. V (1998) Interictal seizure resections show two configurations of endothelial Glut1 glucose transporter in the human blood-brain barrier. J. Cereb. Blood Flow Metab. 18, 26–42.
Liwnicz, B. H., Leach, J. L., Yeh, M. S., and Privatera, M. (1990) Pericyte degeneration and thickening of basement membranes of cerebral micro vessels in complex partial seizures: electron microscopic study of surgically removed tissue. Neurosurgery 26, 409–420.
Takata, K., Kasahara, T, Kasahara, M., Ezaki, O., and Hirano, H. (1991) Localization of Na+-dependent active type and erythrocyte/HepG2-type glucose transporters in rat kidney: immunofluorescence and immunogold study. J. Histochem. Cytochem. 39, 287–298.
Bendayan, M., Roth., J., Perrelet, A., and Orci, L. (1980) Quantitative immunocytochemical localisation of pancreatic secretory proteins of the rat acinar cell. J. Histochem. Cytochem. 28,149–160.
Craig, S. and Goodchild, D. J. (1982) Postembedding immunolabelling. Some effects of tissue preparation on he antigenicity of plant proteins. Eur. J. Cell Biol. 28, 251–256.
Cornford, E. M., Hyman, S., and Pardridge, W. M. (1993) An electron microscopic immunogold analysis of developmental upregulation of the blood-brain barrier GLUT1 glucose transporter. J. Cereb. Blood Flow Metab. 663, 7–18.
Cornford, E. M., Hyman, S., Cornford, M. E., Damian, R. T., and Raliegh, M. J. (1998) A single glucose transporter configuration in normal primate brain endothelium: Comparison with resected human brain. J. Neuropath. Expl. Neurol. 57, 699–713.
Wolff, J. R., and Bar, T. (1972) “Seamless” endothelia in brain capillaries during development of the rat’s cerebral cortex. Brain Res. 41,17–24.
Brightman, M.W., and Kaya, M. (2000) Permeable endothelium and the interstitial space of brain Cell. Mol. Neurobiol. 20,111–130.
Kaya, M., Chang, L., Truong, A., and Brightman, M. W. (1996) Chemical induction of fenestrae in vessels of the blood brain barrier. Exp. Neurol. 142, 6–13.
Hashizume, K., and Black, K. L. (2002) Increased endothelial vesicular transport correlates with increased blood-tumor barrier permeability induced by bradykinin and leukotriene C4. J. Neuropath. Expl. Neurol. 61, 725–735.
Fischbarg, J., Kuang, K. Y., Hirsch, J., Lecuona, S., Rogozuiaski, L., and Silverstein, S. C. (1989) Evidence that the glucose transporter serves as a water channel. Proc. Natl. Acad. Sci. USA 86, 8397–8401.
Fischbarg, J., Kuang, K. Y., Vera, J. C, Arant, S., Silverstein, S. C, Loike, J., and Rosen, O. M. (1990) Glucose transporters serve as water channels. Proc. Natl. Acad. Sci. USA 87, 3244–3247.
Loike, J. D., Cao, L., Kuang, K., Vera, J. C, Silverstein, S. C, and Fischbarg, J. (1993) Role of facilitative glucose transporters in diffusional water permeability through J744 cells. J. Gen. Physiol. 102, 897–906.
Fischbarg, J., and Vera, J. C. (1995) Multifunctional transporter models: Lesson from the transport of water sugars and ring compounds by GLUTs. Amer. J. Physiol. 268, C1077–C1089.
Pappenheimer, J. R., and Setchell, B. P. (1973) Cerebral glucose transport and oxygen consumption in sheep and rabbits. J. Physiol. London 233, 529–551.
Pardridge, W. M., and Oldendorf, W. H. (1975) Kinetics of blood-brain barrier transport of hexoses. Biochim. Biophys. Acta 382, 377–392.
Gjedde, A., and Christensen, O. (1984) Estimates of Michaelis-Menten constants for the two membranes of the brain endothelium. J. Cereb. Blood Flow Metab. 4, 241–249.
Cunningham, V J., Hargreaves, R. J., Pelling, D., and Moorhouse, S. R, (1986) Regional blood-brain glucose transfer in the rat: A novel double-membrane kinetic analysis. J. Cereb. Blood Flow Metab. 6, 305–314.
Hargreaves, R. J., Planas, A. M., Cremer, J. E., and Cunningham, V J. (1986) Studies on the relationship between cerebral glucose transport and phosphorylation using 2-deoxyglucose. J. Cereb. Blood Flow Metabol. 6, 708–716.
Cremer, J. E., Seville, M. P., and Cunningham, V J. (1988) Tracer 2-deoxyglucose kinetics in brain regions of rats given kainic acid. J. Cereb. Blood Flow Metabol. 8, 244–253.
Bolz, S., Farrell, C. L., Dietz, K., and Wolburg, H. (1996) Subcellular distribution of glucose transporter (GLUT1) during development of the blood brain barrier in rats. Cell Tissue Res. 284, 355–365.
Gerhart, D. Z., LeVasseur, R. J., Broderius, M. A., and Drewes, L. R. (1989) Glucose transporter localization in brain using light and electron immunocytochemistry. J. Neurosci. Res. 22, 464–472.
Cornford, E. M., Hyman, S., Cornford, M. E., and Caron, M. J. (1996) Glut1 glucose transporter activity in human brain injury. J. Neurotrauma 13, 523–536.
Kumagai, A. K., Vinores, S. A., and Pardridge, W. M. (1996) Pathological upregulation of inner blood-retinal barrier Glut1 glucose transporter expression in diabetes mellitus. Brain Res. 706, 313–317.
Vorbrodt, A. W., Dobrogowska, D. H., Ueno, M., and Tarnawski, M. (1995) A quantitative immunocytochemical study of blood-brain barrier to endogenous albumin in cerebral cortex and hippocampus of senescence-accelerated mice (SAM). Folia Histochem. Cytobiol. 33, 229–237.
Vorbrodt, A. W., Dobrogowska, D. H., Tarnawski, M., and Lossinski, A. S. (1994) A quantitative immunocytochemical study of the osmotic opening of the blood-brain barrier to endogenous albumin. J. Neurocytol. 23, 772–800.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2003 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Cornford, E.M., Hyman, S., Cornford, M.E. (2003). Immunogold Detection of Microvascular Proteins in the Compromised Blood-Brain Barrier. In: Nag, S. (eds) The Blood-Brain Barrier. Methods in Molecular Medicine™, vol 89. Humana Press. https://doi.org/10.1385/1-59259-419-0:161
Download citation
DOI: https://doi.org/10.1385/1-59259-419-0:161
Publisher Name: Humana Press
Print ISBN: 978-1-58829-073-1
Online ISBN: 978-1-59259-419-1
eBook Packages: Springer Protocols