Mixed Multiplex Staining: Automated RNAscope™ and OPAL™ for Multiple Targets

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.

The ability to simultaneously visualize expression of multiple antigens in cells and tissues can provide powerful insights into cellular and organismal biology. However, standard methods are limited to the use of just two or three simultaneous probes and have not been widely adopted for routine use in paraffin-embedded tissue. We have developed a novel approach called sequential immunoperoxidase labeling and erasing (SIMPLE) that enables the simultaneous visualization of at least five markers within a single tissue section. Utilizing the alcohol-soluble peroxidase substrate 3-amino-9-ethylcarbazole, combined with a rapid non-destructive method for antibody-antigen dissociation, we demonstrate the ability to erase the results of a single immunohistochemical stain while preserving tissue antigenicity for repeated rounds of labeling. SIMPLE is greatly facilitated by the use of a whole-slide scanner, which can capture the results of each sequential stain without any information loss.

The method described in this paper is available for removal of antibodies retained by tissue antigen after immunohistochemical staining. Its application to the antehypophysis has allowed the successive or the simultaneous localization of two different hormones.