Abstract
Immunohistochemistry (IHC) and immunofluorescence (IF) involve the binding of an antibody to a cellular or tissue antigen of interest and then visualisation of the bound product by fluorescence/with the 3,3′-diaminobenzidine (DAB) chromogen detection system. With increasing numbers of available antibodies against cellular epitopes, IHC and IF are very useful diagnostic tools as well as a means to guide specific therapies that target a particular antigen on cell/tissue samples.
There are several IHC and IF staining methods that can be employed depending on the type of specimen under study, the degree of sensitivity required, and the cost considerations. The following is a basic “generic” method for localising proteins and other antigens by direct, indirect, IHC and IF. The method relies on proper fixation of tissue/cells to retain cellular distribution of antigen and to preserve cellular morphology. Details of reagents required are outlined. Consideration is also given to artefacts and other potential pitfalls and thus means to avoid them.
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References
Coons, A.H., Leduc, E.H., and Connolly, J.M. (1955) Studies on antibody production. I. A method for the histochemical demonstration of specific antibody and its application to a study of the hyperimmune rabbit. J Exp Med. 102 49–60.
Kaplan, M.E., Coons, A.H., and Deane, H.W. (1950) Localization of antigen in tissue cells; cellular distribution of pneumococcal polysaccharides types II and III in the mouse. J Exp Med. 91, 15–30.
Nakane, P.K., and Pierce, G.B. (1966) Jr. Enzyme-labeled antibodies: preparation and application for the localization of antigens. J Histochem Cytochem. 14, 929–31.
Avrameas, S., and Uriel, J. [Method of antigen and antibody labelling with enzymes and its immunodiffusion application]. (1966) C R Acad Sci Hebd Seances Acad Sci D. 262, 2543–2545.
Mason, D.Y., and Sammons, R. (1978) Alkaline phosphatase and peroxidase for double immunoenzymatic labelling of cellular constituents. J Clin Pathol. 31, 454–60.
Faulk, W.P., and Taylor, G.M. (1971) An immunocolloid method for the electron microscope. Immunochemistry. 8, 1081–3.
Maderspach, K., Nemeth K., Simon, J., Benyhe, S., Szucs, M., and Wollemann, M.A. (1991) Monoclonal antibody recognizing kappa- but not mu- and delta-opioid receptors. J Neurochem. 56, 1897–904.
Pichon, J., Hirn, M., Muller, J.M., Mangeat, P., Marvaldi, J. (1983) Anti-cell surface monoclonal antibodies which antagonize the action of VIP in a human adenocarcinoma cell line (HT 29 cells). Embo J. 2, 1017–22.
Somogyi, P., Smith, A.D., Nunzi, M.G., Gorio, A., Takagi, H., and Wu, J.Y. (1983) Glutamate decarboxylase immunoreactivity in the hippocampus of the cat: distribution of immunoreactive synaptic terminals with special reference to the axon initial segment of pyramidal neurons. J Neurosci. 3, 1450–68.
Stefanini, M., De Martino, C., and Zamboni, L. (1967) Fixation of ejaculated spermatozoa for electron microscopy. Nature. 216, 173–4.
Melan, M., and Sluder, G. (1992) Redistribution and differential extraction of soluble protein in permeabilized cultured cells. J Cell Sci. 101, 731–743.
Hannah, M., Weiss, U. and Huttner, W. (1998) Differential extraction of protein from paraformaldehyde fixed cells: Lessons from synaptophysin and other membrane proteins, Methods. 16, 170–181.
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Katikireddy, K.R., O’Sullivan, F. (2011). Immunohistochemical and Immunofluorescence Procedures for Protein Analysis. In: O'Driscoll, L. (eds) Gene Expression Profiling. Methods in Molecular Biology, vol 784. Humana Press. https://doi.org/10.1007/978-1-61779-289-2_11
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DOI: https://doi.org/10.1007/978-1-61779-289-2_11
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