Confocal microscopy for intracellular co-localization of proteins

Confocal laser scanning microscopy is the best method to visualize intracellular co-localization of proteins in intact cells. Because of the point scan/pinhole detection system, light contribution from the neighborhood of the scanning spot in the specimen can be eliminated, allowing high Z-axis resolution. Fluorescence detection by sensitive photomultiplier tubes allows the usage of filters with a narrow bandpath, resulting in minimal cross-talk (overlap) between two spectra. This is particularly important in demonstrating co-localization of proteins with multicolor labeling. Here I describe the methods outlining the detection of transiently expressed tagged proteins and the detection of endogenous proteins. Ideally, the intracellular co-localization of the two endogenous proteins should be demonstrated. However, when antibodies raised against the protein of interest are unavailable for immunofluorescence, or the available cell lines do not express the protein of interest sufficiently enough for immunofluorescence, an alternative method is to transfect cells with expression plasmids that encode tagged proteins and stain the cells with anti-tag antibodies after transient transfection. However, it should be noted that the tagging of proteins of interest or their overexpression could potentially alter the intracellular localization or the function of the target protein.