A technique for demonstrating the presence of a specific type of MOLECULE in tissue using antibodies produced specifically for them. ANTIBODIES are produced (or raised, to use the technical term) by sensitizing an animal to a specific molecule (or part of a molecule); that animal will then develop antibodies to that molecule which can be extracted from its BLOOD and purified. The raised antibodies may be polyclonal or MONOCLONAL ANTIBODIES.
In order to do immunohistochemistry one takes sections of tissue and incubates them with a primary antibody. This will produce, within the tissue, ANTIGEN-antibody complexes. These can be visualized in a number of ways. The complex can be tagged with a fluorescent marker (a FLUOROCHROME—a chemical that emits light [fluoresces] when light of a specific wavelength is shone on to it) which can then be detected using a fluorescent microscope. This procedure is known as IMMUNOFLUORESCENCE, and is not commonly used. A more stable visualization technique is to bind diaminobenzidene (DAB) to the antigen-antibody complex using peroxidase. This produces a stable reaction product which is coloured and can be detected using normal light microscopy.
It may be the case though that this reaction is difficult to detect. If so, the signal can be increased by the addition of what are essentially further layers of antibody. This is achieved using a secondary antibody which works as follows. If the primary antibody was raised in, for instance, a mouse, an appropriate secondary antibody would be an anti-mouse IGG (IgG being one of the principal types of antibody) raised in another species (goat or sheep for example). This will then bind to the primary antibody, which it will treat as an antigen. Peroxidase-antiperoxidase (PAP) complex to mouse can then be used to bind to the anti-mouse IgG. What this procedure will have done is to increase dramatically the number of sites at which DAB can be bound, but all of the binding is still dependent on the original interaction of the primary antibody with its antigen. This procedure is known as signal amplification: the PAP technique is just one of several that can be used; others include the avidin-biotin complex (ABC) and tyramide signal amplification.
Immunohistochemistry has been an astonishingly successful technique, allowing neuroanatomists to demonstrate the presence in brain of specific molecules. This of course is one of the central techniques of CHEMICAL NEUROANATOMY. An interesting problem that remains is the quantification of immunohistochemically treated material.
