Immunohistochemical signal amplification by catalyzed reporter deposition and its application in double immunostaining

The biotinyl-tyramide substrate of the horseradish peroxidase Enzyme has been recently introduced to amplify immunohistochemical signals. We applied either fluorochromeor biotin-conjugated tyramine to improve the detection of different antigens in sections of rat stomach, pancreas, and hypothalamus. A ten- to 100-fold increase in staining efficiency was achieved, depending on the antibody, with either fluorescent or peroxidase detection systems. The amplification method was particularly useful for increasing a weak signal of conventional immunostaining caused by suboptimal tissue fixation. At a very low concentration of the primary antibody, the antigen can no longer be detected by a conventional fluorescent secondary antibody but is still detectable after amplification. When an antibody is used at this very low concentration and is detected by a fluorescent amplification method, another primary antibody, raised in the same host species, can be used and demonstrated with a different fluorochrome in subsequent conventional immunostaining of the same section. In this way it becomes possible to immunostain the same section with two different primary Antibodies raised in the same host species. Samples for such double immunostaining are demonstrated here using pairs of monoclonal Antibodies (to tyrosine hydroxylase and oxytocin) in the hypothalamus and polyclonal Antibodies (to glucagon and neurofilament M) in sections of rat pancreas. Because in many cases the availability of Antibodies is limited, the amplification method can be a quick and efficient tool for double immunostaining with Antibodies from the same host species.