FIGURE 1.5 • Direct and indirect immunoﬂuorescence. a. In direct immunofluorescence, a fluorochrome-labeled primary antibody reacts with a specific antigen within the tissue sample. Labeled structures are then observed in the fluorescence microscope in which an excitation wavelength (usually ultraviolet light) triggers the emission of another wavelength. The length of this wavelength depends on the nature of the fluorochrome used for antibody labeling. b. The indirect method involves two processes. First, the specific primary antibodies react with the antigen of interest. Second, the secondary antibodies, which are fluorochrome labeled, react with the primary antibodies. The visualization of labeled structures within the tissue is the same in both methods and requires the fluorescence microscope.
FIGURE 1.6 • 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 nucleation 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– -tubulin and anti– -tubulin monoclonal antibodies (primary antibodies) and visualized by secondary antibodies conjugated with fluorescein dye (fluorescein isothiocyanate–goat anti-mouse immunoglobulin G). The antigen–antibody reaction, performed directly on the glass coverslip, results 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 m in a uniform radial array. 1,400. (Photomicrograph courtesy of Drs. Wilma L. Lingle and Vivian A. Negron.) (e.g., horseradish peroxidase), that convert colorless substrates into an insoluble product of a specific color that precipitates at the site of the enzymatic reaction. The staining that results from this immunoperoxidase method can be observed in the light microscope (Folder 1.4) with either direct or indirect immunocytochemical methods. In another variation, colloidal gold or ferritin (an iron-containing molecule) can be attached to the antibody molecule. These electron-dense markers can be visualized directly with the electron microscope.
Hybridization is a method of localizing messenger RNA (mRNA) or DNA by hybridizing the sequence of interest to a complementary strand of a nucleotide probe.
In general, the term hybridization describes the ability of single-stranded RNA or DNA molecules to interact (hybridize) with complementary sequences. In the laboratory, hybridization requires the isolation of DNA or RNA, which is then mixed with a complementary nucleotide sequence (called a nucleotide probe). Hybrids are detected most often using a radioactive label attached to one component of the hybrid.
Binding of the probe and sequence can take place in a solution or on a nitrocellulose membrane. In in situ hybridization, the binding of the nucleotide probe to the DNA or RNA sequence of interest is performed within cells or tissues, such as cultured cells or whole embryos. This technique allows the localization of specific nucleotide sequences as small as 10 to 20 copies of mRNA or DNA per cell.