IMMUNOFLUORESCENCE SIGNAL AMPLIFICATION BY THE ENZYME-CATALYZED DEPOSITION OF A FLUORESCENT REPORTER SUBSTRATE (CARD)

Progress has been made in improving the immunohistochemical detection of antigens for imaging and flow cytometry. We report the synthesis of a novel fluorescent horseradish peroxidase substrate, Cy3.29-tyramide , and its application in an enzyme-based signal amplification system, catalyzed reporter deposition (CARD). The catalyzed deposition of Cy3. 29-tyramide was used to detect cell surface markers such as CD8 and CD 25 on tonsil tissue and human lymphocytes. We compared the fluorescenc e CARD method to standard indirect immunofluorescence detection method s and found that an amplification of up to 15-fold was possible with C ARD. The detection of the intracellular protein myosin II in fibroblas tic cells and rabbit serum proteins blotted onto nitrocellulose was al so improved. Thus, fluorescent CARD is a simple modification that can be made to standard immunofluorescence staining protocols to enhance s ignificantly the detection of antigens. (C) 1996 Wiley-Liss, Inc.