To localize enzymes in tissue sections, special care must be taken in fixation to preserve the enzyme activity. Usually, mild aldehyde fixation is the preferred method. In these procedures, the reaction product of the enzyme activity, rather than the enzyme itself, is visualized. In general, a capture reagent, either a dye or a heavy metal, is used to trap or bind the reaction product of the enzyme by precipitation at the site
FIGURE 1.3 • Electron histochemical procedure for localization of membrane ATPase in epithelial cells of rabbit gallbladder.
Dark areas visible on the electron micrograph show the location of the enzyme ATPase. This enzyme is detected in the plasma membrane at the lateral domains of epithelial cells, which correspond to the location of sodium pumps. These epithelial cells are involved in active transport of molecules across the plasma membrane. 26,000.
light of different wavelengths (e.g., ultraviolet light) and then emit visible light of a specific wavelength (e.g., green, yellow, red). Fluorescein, the most commonly used dye, absorbs ultraviolet light and emits green light. Antibodies conjugated with fluorescein can be applied to sections of lightly fixed or frozen tissues on glass slides to localize an antigen in cells and tissues. The reaction of antibody with antigen can then be examined and photographed with a fluorescence microscope or confocal microscope that produces a three-dimensional reconstruction of the examined tissue (Fig. 1.4).
Two types of antibodies are used in immunocytochemistry: polyclonal antibodies that are produced by immunized animals and monoclonal antibodies that are produced by immortalized (continuously replicating) antibody-producing cell lines.
In a typical procedure, a specific protein, such as actin, is isolated from a muscle cell of one species, such as a rat, and injected into the circulation of another species, such as a rabbit. In the immunized rabbit, the rat’s actin molecules are recognized by the rabbit immune system as a foreign antigen. This recognition triggers a cascade of immunologic reactions involving multiple groups (clones) of immune cells called B lymphocytes. The cloning of B lymphocytes eventually leads to the production of anti-actin antibodies. Collectively, these polyclonal antibodies represent mixtures of different antibodies produced by many clones of B lymphocytes that
FIGURE 1.4 • Confocal microscopy image of a rat cardiac muscle cell. This image was obtained from the confocal microscope using the indirect immunofluorescence method. Two primary antibodies were used. The first primary antibody recognizes a specific lactate transporter (MCT1) and is detected with a secondary antibody conjugated with rhodamine (red). The second primary antibody is directed against the transmembrane protein CD147, which is tightly associated with MCT1. This antibody was detected by a secondary antibody labeled with fluorescein (green). The yellow color is visible at the point at which the two labeled secondary antibodies exactly co-localize within the cardiac muscle cell. This three-dimensional image shows that both proteins are distributed on the surface of the muscle cell, whereas the lactate transporter alone is visible deep to the plasma membrane. (Courtesy of Drs. Andrew P. Halestrap and Catherine Heddle.) each recognize different regions of the actin molecule. The antibodies are then removed from the blood, purified, and conjugated with a fluorescent dye. They can now be used to locate actin molecules in rat tissues or cells. If actin is present in a cell or tissue, such as a fibroblast in connective tissue, then the fluorescein-labeled antibody binds to it and the reaction is visualized by fluorescence microscopy.