IMMUNOHISTOCHEMICAL SIGNAL AMPLIFICATION BY CATALYZED REPORTER DEPOSITION AND ITS APPLICATION IN DOUBLE IMMUNOSTAINING

The biotinyl-tyramide substrate of the horseradish peroxidase enzyme h as been recently introduced to amplify immunohistochemical signals. We applied either fluorochrome- or biotin-conjugated tyramine to improve the detection of different antigens in sections of rat stomach, pancr eas, and hypothalamus, A ten- to 100-fold increase in staining efficie ncy was achieved, depending on the antibody, with either fluorescent o r peroxidase detection systems, The amplification method was particula rly 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 conve ntional fluorescent secondary antibody but is still detectable after a mplification, 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 demonstrat ed with a different fluorochrome in subsequent conventional immunostai ning of the same section, In this way it becomes possible to immunosta in the same section with two different primary antibodies raised in th e same host species, Samples for such double immunostaining are demons trated here using pairs of monoclonal antibodies (to tyrosine hydroxyl ase and oxytocin) in the hypothalamus and polyclonal antibodies (to gl ucagon and neurofilament M) in sections of rat pancreas, Because in ma ny cases the availability of antibodies is limited, the amplification method can be a guide and efficient tool for double immunostaining wit h antibodies from the same host species.