Simultaneous Visualization of Multiple Antigens with Tyramide Signal Amplification using Antibodies from the same Species

TLDR

Immunohistochemistry has prompted efforts to stain multiple molecules in a single tissue section, but this is difficult because most primary antibodies are rabbit‑derived, making it hard to distinguish signals from two rabbit antibodies, and existing protocols have serious limitations. The authors present a simple, rapid, and inexpensive method to overcome this limitation. The method uses same‑host antibodies and tyramide signal amplification to visualize multiple antigens with commercial fluorescent dyes, enabling detection of both rare and abundant targets. The technique offers two major advantages over existing methods.

Abstract

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.