VISUALIZING CELL COMPONENTS USING ANTIBODIES
Antibodies can be used to visualize the location of specific proteins within the cell. Immunocytochemistry refers to the visualization of specific antigens in cultured cells, whereas immunohistochemistry refers to their visualization in prepared tissue sections. In either technique, the first step is to prepare the cells. They must be treated to maintain their cellular architecture, so that the cells appear much as they would if still alive. Usually, the cells are treated with crosslinking agents such as formaldehyde or with denaturants like acetone or methanol. In immunohistochemistry, tissue samples can be frozen and then sliced into small thin sections (about 4 mm), providing a two-dimensional view of the cell. Another option is to embed the tissue sample in paraffin wax. Here the cells are first dehydrated in a series of alcohol solutions, and then treated with the wax. The tissue is then sectioned into thin two-dimensional slices as for frozen tissues.
Preserved cells then need to be permeabilized so that the antibody can enter. Once a single, thin layer of prepared cells or tissue sections is readied, the cells are treated to make the antigen more accessible to the antibody. If in wax, the tissue sections are dewaxed and rehydrated in solution. Fixed tissue sections can be irradiated with microwaves, which break the crosslinks induced by the fixative, or the samples can be heated under pressure. After permeabilization, the primary antibody finds its antigen within the sample and binds. A secondary antibody contains the detection system. The secondary antibody binds to the primary antibody/antigen complex, and then the appropriate reagents are added to visualize the location of the complex. (In some cases, a single antibody, with an attached detection system, is used.)
The antibody is detected using enzymes or by fluorescent labels. A common enzyme-mediated detection system is alkaline phosphatase, as with the ELISA—see Fig. 6.15. Fluorescently labeled antibodies must be excited with UV light, on which the fluorescent label emits light at a longer wavelength. Samples are directly visualized with a microscope attached to a UV light source (Fig. 6.17). Fluorescent antibodies tend to bleach out when exposed to excess UV; therefore, the microscope is attached to a camera to record the data as a digital image.