Figure

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 lateral domains of epithelial cells, which corresponds to the location of sodium pumps. These epithelial cells are involved in active transport of molecules across the plasma membrane. x26,000.

the fluorescent dye bound to the antibodies (Fig. 1.4). A fluorescence microscope is used to display the fluorescein now attached indirectly to the antigen. It is also possible to conjugate substances such as gold or ferritin (an iron-containing molecule) to the antibody molecule. These markers can be visualized directly with the EM.

Enzyme histochemical methods are combined with traditional immunocytochemical methods to amplify the localization reaction between an antigen and an antibody

In these methods, horseradish peroxidase enzyme is conjugated with a primary antibody. Following the antigen-antibody reaction, the histochemical procedure for demonstrating peroxidase activity is run to reveal the location of the antigen-antibody complex (direct reaction). A further refinement of this method attaches the peroxidase to an anti-y-globulin (secondary antibody) that binds to the primary antibody, further amplifying the reaction (indirect reaction). Because the end product of the peroxidase reaction is also visible in the EM, this method is easily adapted to EM immunocytochemistry. Monoclonal antibodies conjugated with ferritin or gold particles may be used as primary antibody stains to achieve even more precise localization of antigens in tis sue sections examined in the TEM than is possible with traditional polyclonal antibodies.

An additional advantage of the indirect labeling method is that a single secondary antibody can be used to localize the intracellular or tissue-specific binding of several different primary antibodies. For light microscopic studies, the secondary antibody can be conjugated with different fluorescent dyes so that multiple labels can be shown in the same tissue section (see page 49).

Autoradiography

Autoradiography makes use of a photographic emulsion placed over a tissue section to localize radioactive material within tissues

Many small molecular precursors of larger molecules, such as the amino acids that make up proteins and the nucleotides that make up nucleic acids, may be tagged by incorporation of a radioactive atom or atoms into their molecular structure. The radioactivity is then traced to localize the larger molecules in cells and tissues. Labeled precursor molecules can be injected into animals or introduced into

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