There are different techniques for performing immunohistochemistry but all are based on the same principle. An antibody, either monoclonal or polyclonal, directed against the antigen under study, is applied to an appropriately processed tissue section, and labeled, so that its binding site can be detected.
In the simplest method a label is directly bound to this (primary) antibody. If a chro-mogenic labeling is preferred, an enzyme (either peroxidase or alkaline phosphatase) is employed with a chromogenic substrate. The enzyme acts on the substrate to convert it into an insoluble pigment that precipitates at the site of the bound antibody, revealing where it is located in the cell or tissue. Fluorescent labels bound to the antibody require fluorescent microscopy with ultraviolet illumination and selected filters in order to be visualized.
With an appropriate counterstaining of the tissue section, the labeled antigen can be discretely localized.
To make the method more versatile and sensitive, different techniques have been employed. In routine use the primary antibody is not labeled directly, but indirectly by using a labeled secondary antibody directed against the constant part (Fc portion) of the primary antibody. In more sensitive methods, tertiary complexes involving more labeling molecules are used, e.g., through a biotin-avidin-mediated link a tertiary complex carrying the chromogen may be formed to label the antigen. In newly developed techniques the reagent contains the secondary antibody directed against the primary antibody with several molecules of the enzyme linked by a polymer "backbone." That enhances the labeling and shortens the staining procedure because the secondary antibody step is omitted.
A chromogenic immunohistochemical labeling is stable and can be readily evaluated under routine light microscopy; no special equipment is needed. Specialized equipment is necessary for the evaluation of immunofluorescent stains, and the staining results are not permanent. This method, although precise and sensitive, is therefore impractical for routine studies of cervical pathology.
Caution must be employed for detecting antigens in sections of formalin-fixed and paraffin-embedded tissues. The epitope of the antigen must be unmasked in most instances, because formalin fixation causes proteins to cross-link, preventing antibodies reacting with the epitope of the antigen.
For further technical details, refer to handbooks on microscopic methods in molecular biology. For various groundbreaking reports on immunohistochemical methods with polyclonal and monoclonal antibodies, refer to the historical literature (Moll et al. 1982,1983; Czernobilsky et al. 1984; Makin et al. 1984; Tsutsumi et al. 1984; Levy et al. 1988).
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