Immunoperoxidase Staining of Gene Products in Cultured Cells Using Monoclonal Antibodies

Antibodies, in general, provide the most sensitive and specific methods for detecting the protein products of genes. Immunoperoxidase techniques described here detect 10³ -10⁵ molecules/cell (depending on whether the protein is dispersed within the cell or concentrated at high density in a particular compartment); with various enhancement methods, this can be improved more than tenfold. Immunohistochemistry is particularly suitable for analyzing transfection in vitro, because it examines individual cells; methods based on analysis of bulk RNA (e.g., Sl nuclease protection [see Chapter 21 ], this volume]) or protein (e.g., immunoblotting) are considerably less sensitive when only a small proportion of cells are transfected. Immunoperoxidase reactions are generally more sensitive than immunofluorescence (Chapter 27) and immunogold methods and require no special optics to observe. Also, the reaction product is stable for years. On the other hand, immunofluorescence is easier to perform than immunoperoxidase, the conjugated antibody is generally more stable and so more reliable, and the fluorescence signal is easier to demonstrate in black-and-white photography. If a good fluorescence microscope is available and the signal is expected to be reasonably strong, then immunofluorescence is the method of choice.