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 CD25 on tonsil tissue and human lymphocytes. We compared the fluorescence CARD method to standard indirect immunofluorescence detection methods and found that an amplification of up to 15-fold was possible with CARD. The detection of the intracellular protein myosin II in fibroblastic cells and rabbit serum proteins blotted onto nitrocellulose was also improved. Thus, fluorescent CARD is a simple modification that can be made to standard immunofluorescence staining protocols to enhance significantly the detection of antigens.
Chao
J
J
Center for Light Microscope Imaging and Biotechnology, Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA.
DeBiasio
R
R
Zhu
Z
Z
Giuliano
K A
KA
Schmidt
B F
BF
D016475:3T3 Cells; D000818:Animals; D000954:Antigens, Surface / Q000032:analysis; D015415:Biomarkers / Q000032:analysis; D002232:Carbocyanines / Q000737:chemistry; D003101:Collodion / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay / Q000295:instrumentation / Q000379:methods*; D005455:Fluorescent Antibody Technique / Q000295:instrumentation / Q000379:methods*; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000378:metabolism*; D006735:Horseradish Peroxidase / Q000737:chemistry; D006801:Humans; D008214:Lymphocytes; D051379:Mice; D015394:Molecular Structure; D024462:Myosin Type II / Q000032:analysis; D015203:Reproducibility of Results; D014439:Tyramine / Q000031:analogs & derivatives / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*
D000954:Antigens, Surface; D015415:Biomarkers; D002232:Carbocyanines; C481082:Cy3.29-tyramide; D005456:Fluorescent Dyes; C089187:cyanine dye 3; D003101:Collodion; D006735:Horseradish Peroxidase; D024462:Myosin Type II; D014439:Tyramine