Immunofluorescence and Immunoelectron Microscopy of Microinjected and Transfected Cultured Cells

Immunofluorescence microscopy is the most common method to analyze expression and localization of a given protein in cells that have been microinjected or transfected previously with the appropriate DNA constructs. It offers the advantage that it is quick, easy to perform, and allows examination of a large number of cells within a short time. However, illumination with UV light is often damaging for the cells, and the fluorescence tends to bleach as a result of the excitation of the fluorochrome by the UV light. Nowadays, these disadvantages have been overcome by sophisticated systems such as video intensification cameras and confocal microscopy. In addition, the information that can be obtained by immunofluorescence microscopy is also restricted by the limited resolution of the optical lens. Moreover, some fixation conditions can induce artifactual changes in the intracellular localization of a significant number of molecules (Melan and Sluder 1992; Griffiths et al. 1993). Therefore, if the precise localization of a protein is being studied, it will always be necessary to look at the fine structure of the cell, using immunoelectron microscopy techniques. Nevertheless, whenever possible, the utilization of immunofluorescence in combination with immunoelectron microscopy is preferable. In this chapter, we describe in detail protocols for immunofluorescence and immunoelectron microscopy analyses which are particularly suited for cells in culture.