Enzyme-Cytochemistry identifies an enzyme within a tissue or cell type by a specific substrate reaction resulting in a visible marker.

Substrate - the substance acted upon by an enzyme.

Trapping agent - binds to the substrate and contains a visible reaction product such as a heavy metal or a colored reaction product.

Enzymes may be classified by their method of demonstration.

A. Simultaneous capture describes the procedure where a reagent in the incubation medium combines with the reaction product. An example of this technique is the diazo method for alkaline phosphatase.

B. Post-incubation coupling is based on the production of an insoluble reaction product which is then coupled with a colored or opaque substance. Rutenburg and Seligman for acid phosphatase is an example.

C. Self-colored substrate reaction is obtained when a water-soluble dye is made insoluble due to the removal of a hydrophilic group, by the enzyme. A colored precipitate is then produced at the site of enzyme activity.

D. Intramolecular rearrangement produces a colored insoluble participate at the sites of enzyme activity of an otherwise colorless substrate.

Enzymatic reactions are often coupled with immunochemical methods.

Immunochemical Methods

Immunocytochemistry or immunohistochemistry is the identification of a tissue constituent (protein, lipopolysaccharide, etc.) in situ by means of a specific antigen/antibody reaction tagged by a visible label. immunohistochemistry usually refers to a light level detection and immunocytochemistry usually refers to detection at EM resolution.

The antigen is the protein or target of interest. An antibody is a structure created by the immune system to seek out and bind with a specific antigen. An antigen is composed of many segments called epitopes. The epitope is the region that will bind with the antibody.

Because of the special relationship between the antigen and antibody, care must be taken to assure that neither is altered beyond recognition by it’s counterpart, while at the same time maintaining the integrity of the tissue or cell in which the antigen resides.

Antibody structure

The overall structure of the antibody is a Y shape. The combining (or reactive) sites are located on the ends of the two arms. The structure of these two ends vary from one antibody to another. The third region is not involved in antigen binding. The reactive sites of the antibody are formed by four polypeptide chains bonded by bridges between sulfur-containing amino acids. A pair of identical light chains is linked to a pair of heavy chains.

Figure 1. Schematic model of immunoglobulin molecule.

Antibody structures are complex multichain proteins that have the same overall shape, yet each has unique regions that promotes binding to one antigen and not another. Folding of the antibody chains create clefts and hollows, which conform to the shape of the antigen, so that weak intermolecular forces can bind the antigen to the antibody.

Types of Antibodies:

A. Monoclonals are antibodies specific for only one antigenic site.

B. Polyclonals are a mixture of antibodies, each specific for particular epitope of the same antigen.

Types of Immuno labeling:

A. The direct labeling method requires only one step in which a specific antibody is complexed directly to the visible marker.

Figure 2. Schematic representation of direct labeling.

B. Indirect labeling is a two-step method which uses an antibody specific for the antigen and a secondary antibody coupled to a visible marker.

Figure 3. Indirect labeling schematic

C. Multiple labeling (also referred to as double or triple labeling, etc.) refers to the simultaneous localization of two or more different antibodies. Obviously a different marker for each antibody must be employed

Specific Markers:

A. Fluorescent probes may be used for obtaining antigen localization at the light level. With the invention of the laser scanning confocal microscope, this technique is especially powerful, and relatively simple.

B. Gold particles are currently the most popular tags for electron microscopy immunostaining and are used with increasing frequency at the light level as well. The particles are discreet in the electron microscope and hard to mistake. They come in a variety of sizes for double labeling. At the light microscope, a silver enhancement technique must be employed for visualization. An epi-polarizing filter enhances detection of the heavy metal label.

C. Horseradish Peroxidase (HRP)is an enzyme tag attached to an immunoglobulin with the intent of using the catalytic properties of the enzyme to form an insoluble reaction product.

D. Other enzyme markers are alkaline phosphatase, beta galactosidase, and glucose oxides. These enzymes, like HRP, require a chromogen (usually diaminobenzidine,DAB or 3-amino-9-ethylcarbazole, AEC).