On Sat, 13 May 2000, Greg Dobbin wrote:

> Could someone provide me with a brief explanation of the tyramide
> amplification technique and perhaps a reference or two, that would
> describe the method.

    The terms "tyramide amplification" and "CARD" (catalysed
    reporter deposition" are a little confusing. The method is
    actually a sensitive technique for detecting peroxidase
    activity, as in the case of horseradish peroxidase 
    deposited in a section as a result of immunohistochemistry 
    or in situ hybridization. It has been most used with
    fluorescent in sit. hyb. (FISH). 

    For example, you can make a biotin-tyramine conjugate
    by reacting tyramine with a succinimide derivative of
    biotin. The tyramine end of the new molecule acts
    rather like the chromogen in an ordinary peroxidase
    method. In the presence of hydrogen peroxide, it is
    oxidized at the sites of peroxidase activity. The
    product of oxidation is unstable (possibly a free 
    radical; chemistry is not fully worked out) and it
    covalently binds, immediately, to the section (possibly
    to tyrosine side-chains of protein). One molecule of
    HRP can catalyse the oxidation of many molecules of
    biotin-tyramine, so tissue-bound biotin piles up at
    sites of enzyme activity. It's covalently bound, so
    it doesn't wash off - even if you remove the original
    antibodies with acid washes. The bound biotin can be
    detected with any form of labelled avidin or streptavidin:
    for example, a fluorescently labelled avidin or an
    HRP-labelled avidin. (A 2nd HRP label would be detected
    in the usual way, with DAB, AEC etc.) 

    Tyramine-fluorochrome conjugates provide an even simpler
    approach. They amount to methods for peroxidase activity
    that generate fluorescent products that cannot be washed
    off the tissue because they are covalently bound.

    Tyramide methods is well suited to procedures with more
    than one fluorescent label applied to the same section.
    Several publications describe these techniques. Hopman
    et al (1998) J Histochem Cytochem 46: 771-777 give
    simple instructions for making your own conjugates of
    tyramine with biotin, digoxigenin and various 
    fluorochromes. Similar instructions appeared also in the
    earliest publications, which were for non-histological
    applications (Bobrow et al., 1989 J Immunol Methods
    125: 279-285, and others).
 
    A few comments on the Histonet listserver in the last
    year or so have indicated that the high sensitivity of
    tyramide amplification methods can carry the price of
    "excessive" staining (of everything). This should be
    avoidable by diluting the reagents or by using a less
    sensitive method for peroxidase activity.

 John A. Kiernan,
 Department of Anatomy & Cell Biology,
 The University of Western Ontario,
 LONDON,  Canada  N6A 5C1