Microtubules visualized by immunocytochemical methods. The behavior of microtubules (elements of the cell cytoskeleton) obtained from human breast tumor cells can be studied in vitro by measuring their nu-cleation activity, which is initiated by the centrosome. This image was photographed in the fluorescence microscope. By use of indirect immunofluorescence techniques, microtubules were labeled with a mixture of anti-a-tubulin and anti—/3-tubuIin monoclonal antibodies (primary antibodies) and visualized by secondary antibodies conjugated with fluorescein dye (fluorescein isothiocyanate-goat antimouse immunoglobulin G). The antigen-antibody reaction, performed directly on the glass coverslip, resulted in visualization of tubulin molecules responsible for the formation of more than 120 microtubules visible on this image. They originate from the centriole and extend outward approximately 20 to 25 ¿im in a uniform radial array, x 1,400. (Photomicrograph courtesy of Dr. Wilma L. Lingle and Ms. Vivian A. Negron.)

cell or organ cultures. In this way, synthesis of DNA and subsequent cell division, synthesis and secretion of proteins by cells, and localization of synthetic products within cells and in the extracellular matrix have been studied.

Sections of specimens that have incorporated radioactive material are mounted on slides. In the dark, the slide is usually dipped in a melted photographic emulsion, thus producing a thin photographic film on the surface of the slide. After appropriate exposure in a light-tight box, usually for days to weeks, the exposed emulsion on the slide is developed by standard photographic techniques and permanently mounted with a coverslip. The slides may be stained either before or after exposure and development. The silver grains in the emulsion over the radioactively la beled molecules are exposed and developed by this procedure and appear as dark grains overlying the site of the radioactive emission when examined with the light microscope (Fig. 1.5a).

These grains may be used simply to indicate the location of a substance, or they may be counted to provide semiquantitative information about the amount of a given substance in a specific location. For instance, after injection of an animal with tritiated thymidine, cells that incorporated this nucleotide into their DNA prior to dividing but that have not yet divided will have approximately twice as many silver grains overlying their nuclei as will cells that have divided after incorporating the labeled nucleotide.

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