Immunofluorescent Reagents: Preparation and Analysis

Abstract

Immunofluorescent staining techniques for detecting cell surface antigens have evolved over the past forty-five years (Coons et al., 1941) to the point where it is now possible to detect up to five cell surface antigens simultaneously (Horan et al., 1986). To a great degree, this precision has been dependent upon advances in three areas. First, instruments such as flow cytometers were created that could analyze cellular immunofluorescence with much greater speed and precision than analysis utilizing fluorescence microscopes. Second, the introduction of monoclonal antibodies as probes to detect cellular antigens provided remarkable specificity and uniformity. Finally, chemical modifications of fluorochrome molecules facilitated simple conjugation and purification procedures as well as produced fluorochromes which exhibited distinct spectral emission characteristics.