Here we intend to introduce only sufficient knowledge of antibodies to explain the issues that contribute to the way they work in our experimental systems and, consequently, also their limitations and potential artifacts. There are numerous excellent reviews and books covering these topics (eg, Refs. This article cannot hope to summarize the vast amount of very detailed literature concerning antibodies, their generation, and their functionality. What Are Antibodies? What Types Are There? How Are They Generated?
Here we hope to alert the investigator and potential referees to the possible pitfalls that can be encountered. Unfortunately, the scientific literature is pervaded by examples of erroneous results using antibodies, particularly in immunohistochemistry. This guide briefly discusses how antibodies are produced, how they function in the context of immunohistochemistry, and what controls and documentation are essential if a result is to be believed. But how much of this should we believe, and what are the minimal controls that still need to be carried out to ensure adequate scientific rigor in our experiments? Referees and journal editors are becoming alarmed by the often superficial way in which antibody specificity is dealt with ( 2– 4). In the postgenomic era of the Internet we are inundated by information from companies offering large numbers of antibodies, mostly against peptides or recombinant proteins, all of which postulate very high specificity combined with rigorous controls. Although the technique of immunocytochemistry has been around for some 50 years ( 1), the methodology itself is still relatively crude, and our understanding of what factors influence specificity and sensitivity is often rudimentary. They combine extremely high precision of identification at the protein level, with high sensitivity, and also localization at a cellular or even a subcellular scale. Antibodies are a valuable and shared resource within the scientific community it is essential therefore that mistakes involving antibodies and their controls are not perpetuated through inadequate reporting in the literature.Īntibodies, particularly for use in immunohistochemistry, represent one of the most powerful tools in modern biological science. A key factor in all of this is that techniques need to be properly documented and especially antibodies and procedures must be adequately described.
In this review, the authors draw on many years of experience to illuminate many of the more common errors and problematic issues in immunohistochemistry, and how these may be avoided. However, this improved sensitivity has also increased the risks of false-positive and false-negative staining and thereby raised the necessity for proper and adequate controls. New techniques of immunohistochemistry and immunofluorescence have improved both the optical resolution of such protein identification as well as its sensitivity, particularly through the use of amplification methodology. For several decades antibodies raised against specific proteins, peptides, or peptide epitopes have proven to be versatile and very powerful tools to demonstrate molecular identity in cells and tissues.
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