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Simultaneous Visualization of Multiple Antigens With Tyramide Signal Amplification Using Antibodies From the Same Species

Zsuzsanna E. Tóth and Éva Mezey

Craniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland (ZET,EM), and Laboratory of Neuromorphology, Department of Anatomy, Histology and Embryology, Semmelweis University and the Hungarian Academy of Sciences, Budapest, Hungary (ZET)

Correspondence to: Zsuzsanna E. Tóth, CSDB, NIDCR, NIH, Bethesda, MD 20892-2190. E-mail: tothzs{at}ana.sote.hu

After immunohistochemistry (IHC) began to be used routinely, a number of investigators worked on methods for staining multiple molecules in the same tissue sections or cells. Achieving this goal was not easy, however. One reason for this is that the majority of primary antibodies used in IHC reactions are raised in rabbits, and recognizing signals from two different rabbit antibodies is not trivial. Thus, all of the protocols described to date have serious limitations. Here we report a simple, quick, and inexpensive solution to the problem. It has two major advantages over existing methods. First, by using antibodies from the same host, two or more antigens can be visualized in the same section with commercially available fluorescent dyes. Second, because the technique relies on signal amplification, both rare and abundant antigens can be detected. (J Histochem Cytochem 55:545–554, 2007)

Key Words: immunohistochemistry • multiple antigens • multiplex detection • tyramide signal amplification

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