Detecting Fluorochrome-Labeled Cells

Abstract

This protocol describes cell staining using fluorochrome-labeled antibodies. The resolution of subcellular structures using fluorochrome-labeled antibodies exceeds that of the transmitted light microscope because of the visualization of an expanding cone of emitted light from the excited fluorochrome in the specimen. The most commonly used fluorochromes are fluorescein and rhodamine. In recent years, a number of advances in the development of fluorochromes have resulted in brighter and longer-lasting dyes with narrow emission spectra. These are available from a number of commercial suppliers. They can be conjugated to anti-immunoglobulin antibodies, Protein A, Protein G, avidin, or streptavidin. These conjugates are available from many commercial sources. Filter sets are commonly available that will permit independent observation of these two fluorochromes in the same sample. The fluorochrome Texas Red is also used for immunofluorescence, and can be detected using the same filter sets as rhodamine.