Conjugation of Fluorochromes to Antibodies

The use of specific antibodies labeled with a fluorescent dye to localize substances in tissues was first devised by A. H. Coons and his associates. At first, the specific antibody itself was labeled and applied to the tissue section to identify the antigenic sites (direct method) (). Later, the more sensitive and versatile indirect method () was introduced. The primary, unlabeled, antibody is applied to the tissue section, and the excess is washed off with buffer. A second, labeled antibody from another species, raised against the IgG of the animal donating the first antibody, is then applied. The primary antigenic site is thus revealed. A major advantage of the indirect method is the enhanced sensitivity. In addition, a labeled secondary antibody can be used to locate any number of primary antibodies raised in the same animal species without the necessity of labeling each primary antibody.