Abstract
Some components of biological systems can be readily identified solely by their unique structure or other intrinsic physical properties which are evident when visualized in brightfield or various types of interference-based light microscopy (LM). However, for the unambiguous identification and localization of most biological molecular or macromolecular elements within a structural framework, some type of staining/labeling must be employed. This is important for a variety of applications including identification of particular tissues, identification of cells and subcellular components/structures, tracking of cells or subcellular components, and co-localization of cells and cellular components on or within tissues or cells. Labeling is also used to provide quantitative comparisons of epitope density, cell numbers, organelle numbers or volume, and a variety of other types of quantitative data. However, considerable caution must be taken when attempting quantitative or even semi-quantitative analyses. Efficiencies of labeling for different epitopes and antibodies or antibody mixtures vary. The exact relationship of color density, particle numbers, or fluorescence intensity (which can fade during observation), to the actual numbers of labeled sites is critical and often not known. These factors often make quantitative estimations or comparisons very risky.
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References
Albrecht, R.M., and Meyer, D.A. 2008. Molecular labeling for correlative microscopy: LM, LVSEM, TEM, EF-TEM, and HVEM. Ch 6, Low Voltage Scanning Electron Microscopy. H. Schatten and J. Pawley (eds) Springer Science, New York, 171–196.
Bruchez, Jr., M., Moronne, M., Gin, P., Weiss, S., Alivisatos, A.P. 1998. Semiconductor nanocrystals as fluorescent biological labels. Science 281:2013–2016.
Chalfie, M., tyu, Y., Euskirchen, G., Ward, W.W., Prasher, D.C. 1994. Green fluorescent protein as a marker for gene expression. Science 263:802–805.
Chan, W.C.W., Nie, S. 1998. Quantum dot bioconjugates for ultrasensitive nonisotopic detection. Science 281:2016–2018.
Criscitiello, M.F. and Flajnik, MF. 2007. Four primordial immunoglobulin light chain isotypes, including lambda and kappa, identified in the most primitive living jawed vertebrates. Eur J Immunol. 37(10):2683–2694.
Cummings, R.D., Etzler, M.E. 2009. Antibodies and lectins in glycan analysis. Ch. 45, Essentials of Glycobiology, Second Edition. A. Varki, R.D. Cummings, J.D. Esko, H.H. Freeze, P. Stanley, C.R Bertozzi, G.W. Hart, M.E. Etzler (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp. 633–648.
Eppell, S.J., Simmons, S.R., Albrecht, R.M., and Marchant, R.E. 1995. Cell surface receptors and proteins on platelet membranes imaged by scanning force microscopy using immunogold contrast enhancement. Biophysical J. 68:671–680.
French, D.L., Laskov, R., Scharff, M.D. 1989. The role of somatic hypermutation in the generation of antibody diversity. Science 244:1152–1157.
Green, N.M. 1963. Avidin 1. The use of [14C]biotin for kinetic studies and for assay. Biochem J, 89:585–591.
Hamers-Casterman, C., Atarhouch, T., Muyldermans, S., Robinson, G., Hamers,C., Bajyana Songa, E., Bendahman, N. and Hamers, R. 1993. Naturally occurring antibodies devoid of light chains. Nature 363:446–448.
Hansen, J.D., Landis, E.E. and Phillips, R.B. 2005. Discovery of a unique Ig heavy-chain isotype (IgT) in rainbow trout: Implications for a distinctive B cell developmental pathway in teleost fish. Proc Natl Acad Sci U S A. 102(19): 6919–6924.
Heim, R., Cubitt, A., and Tsien, R. 1995. Improved green fluorescence. Nature 373 (6516): 663–664.
Heim, R., and Tsien, R.Y. 1996. Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr Biol 6:178–182.
Horsfall, A.C., Hay, F.C., Soltys, A.J., Jones, M.G. 1991. Epitope mapping. Immunol Today 12:211–213.
Huston, J.S., Levinson, D., Mudgett-Hunter, M., Tai, M-S., Novotny, J., Maroglies, M.N., Ridge, R.J., bruccoleri, R.E., Haber, E., Crea, R., Oppermann, H. 1988. Protein engineering of anitbody binding sites: Recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc Natl Acad Sci U S A. 85:5879–5883.
Kandela, I.K., Bleher, R., and Albrecht, R.M. 2007. Multiple correlative immunolabeling for light and electron microscopy using fluorophores and colloidal metal particles. J Histochem Cytochem 55(10):983–990.
Kandela, I.K., and Albrecht, R.M. 2007. Fluorescence quenching by colloidal heavy metal nanoparticles: Implications for correlative fluorescence and electron microscopic studies. Scanning 29:152–161.
Kandela, I.K., Bleher, R., and Albrecht, R.M. 2008. Correlative light and electron microscopy imunolabeling on ultrathin cryosections of skeletal muscle tissue. Microsc Microanal 14:159–165.
Kindt, T.J., Goldsby, R.A., and Osborn, B.A. eds., 2007. Kuby Immunology, 6th edition, WH Freeman and Company, New York, 2007.
Linscotts Directory of Immunological and Biological Reagents. 2010 Linscotts USA. https://www.linscottsdirectory.com.
Lubeck, M.D., Steplewski, Z., Baglia, F., Klein, M.H., Dorrington, K.J., and Koprowski, H. 1985. The interaction of murine IgG subclass proteins with human monocyte Fc receptors. J. Immunol 135:1299–1304.
McCafferty, J., Griffiths, A.D., Winter, G., Chiswell, D.J. 1990. Phage antibodies: filamentous phage displaying antibody variable domains, Nature 348:552–554.
Nilson, B.H.K., Lögdberg, L., Kastern, W., Björck, L., Åkerström, B. 1993. Purification of antibodies using protein L-binding framework structures in the light chain variable domain, J Immunol Methods, 164:33–40.
Reiter, Y., Brinkmann, U., Jung, S-H., Lee, B., Kasprysyk, P.G., King, C.R., and Pastan, I. 1994. Improved binding and antitumor activity of a recombinant anti-erbB2 immunotoxin by disulfide stabilization of the Fv fragment. J Biol Chem 269:18327–18331.
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Albrecht, R.M., Oliver, J.A. (2011). Labeling Considerations for Confocal Microscopy. In: Price, R., Jerome, W. (eds) Basic Confocal Microscopy. Springer, New York, NY. https://doi.org/10.1007/978-0-387-78175-4_5
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DOI: https://doi.org/10.1007/978-0-387-78175-4_5
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