Immunohistochemistry (IHC) is a common analysis in scientific preclinical research and a precious resource in pathology for morphologic diagnosis and for studying the pathogenesis of diseases. So, the correct methodology and interpretation of an immunohistochemical examination are crucial. A huge challenge in IHC is to avoid artifacts such as non-specific interactions and high background without, at the same time, impairing the antibody-antigen binding.
Good immunohistochemical staining is accomplished when the right amount of primary antibody penetrates the sample to bind its related antigenic target with high specificity. However it takes a deep scientific knowledge of the technique, the tissue sample, and the target protein to discriminate false positives.
In fact, in many cases, the same physiochemical forces that govern specific antibody-antigen interactions, such as ionic interactions, hydrophobic interactions, and hydrogen binding, also contribute to unpleasant artifacts.
There is Nothing More Deceptive Than an Obvious Fact
Commons artifacts in IHC include high background, non-specific binding, overstaining or weak staining.
High background (or overstaining) happens when the level of background becomes so high that it obscures important structures and features of the tissue. The binding of the antibody, either the primary or the secondary antibody, to something else in the tissue than its designated target, such as other proteins or different epitopes in the target protein is referred to as nonspecific binding. Weak staining could be determined as staining of moderate intensity present in 10 % or less of cells.
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