Abstract
In order to test tissue specimens with antibody, they first have to be preserved in fixative, embedded in paraffin, and sectioned very thinly onto glass microscope slides. Any piece of tissue, immediately after excision, must be placed into an adequate volume of fixative. Fixatives vary, but the standard one is 10% buffered formalin. After an optimum fixation time (for formalin, about 16 h), the sample must be embedded in paraffin and sectioned on a microtome. Paraffin-embedded sections placed on positively charged slides (either coated or commercially prepared) are then ready for various pretreatment steps. First, the paraffin must be replaced with water through a series of rehydration steps. Then, depending on the antigen to be tested, the section can be proteolytically digested with enzymes or heat-treated in low or high pH solutions. Following that, the endogenous peroxidase enzyme or oxidative compounds can be quenched in a hydrogen peroxide solution. The sections are then ready to be tested with antibody after an incubation in a normal serum solution blocks any available charged sites.
The opinions or assertions herein represent the personal views of the author and are not to be construed as official or as representing the views of the Department of the Army or the Department of Defense.
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References
Guntern R, Vallet P, Bouras C, Constantinidis J (1989) An improved immunohistostaining procedure for peptides in human brain. Experientia 45:159–161
Battifora H (1991) Assessment of antigen damage in immunohistochemistry. Am J Clin Pathol 96:669–671
Login G, Schnitt S, Dvorak A (1987) Rapid microwave fixation of human tissues for light microscopic immunoperoxidase identification of diagnostically useful antigens. Lab Invest 57:585–591
Baumgartner W, Dettinger H, Schmeer N, Hoffmeister E (1988) Evaluation of different fixatives and treatments for immunohistochemical demonstration of Coxiella burnetii in paraffin-embedded tissues. J Clin Microbiol 26:2044–2047
Ambrogi LP (ed) (1957) Manual of histologic and special staining techniques. Armed Forces Institute of Pathology, Washington, DC
Bratthauer GL, Moinfar F, Stamatakos MD, Mezzetti TP, Shekitka KM, Man YG, Tavassoli FA (2002) Combined E-cadherin and high molecular weight cytokeratin immunoÂprofile differentiates lobular, ductal, and hybrid mammary intraepithelial neoplasias. Hum Pathol 33:620–627
Taschini P, MacDonald D (1987) Protease digestion step in immunohistochemical procedures: ficin as a substitute for trypsin. Lab Med 18:532–536
Stirling J (1990) Immuno- and affinity probes for electron microscopy: a review of labeling and preparation techniques. J Histochem Cytochem 38:145–157
Kitamoto T, Ogomori K, Tateishi J, Prusiner S (1987) Formic acid pretreatment enhances immunostaining of cerebral and systemic amyloids. Lab Invest 57:230–236
Shi S, Key M, Kalra K (1991) Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistoÂchemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem 39:741–748
Tacha DE, Chen T (1994) Modified antigen retrieval procedure: calibration technique for microwave ovens. J Histotechnol 17:365
Man Y-g, Tavassoli FA (1996) A simple epitope retrieval method without the use of microwave oven or enzyme digestion. Appl ImmunoÂhistochem 4:139–141
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Bratthauer, G.L. (2010). Processing of Tissue Specimens. In: Oliver, C., Jamur, M. (eds) Immunocytochemical Methods and Protocols. Methods in Molecular Biology, vol 588. Humana Press. https://doi.org/10.1007/978-1-59745-324-0_13
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DOI: https://doi.org/10.1007/978-1-59745-324-0_13
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