Pearls and pitfalls in neural CGRP immunohistochemistry

This review outlines the pearls and pitfalls of calcitonin-gene related protein (CGRP) immunohistochemistry of the brain. Pearls: In 1985, CGRP was first described in cerebral arteries using immunohistochemistry. Since then, cerebral CGRP (and, using novel antibodies, its receptor components) has been widely scrutinized. Here, we describe the distribution of cerebral CGRP and pay special attention to the surprising reliability of results over time. Pitfalls: Pitfalls might include a fixation procedure, antibody clone and dilution, and interpretation of results. Standardization of staining protocols and true quantitative methods are lacking. The use of computerized image analysis has led us to believe that our examination is objective. However, in the steps of performing such an analysis, we make subjective choices. By pointing out these pitfalls, we aim to further improve immunohistochemical quality. Recommendations: Having a clear picture of the tissue/cell morphology is a necessity. A primary morphological evaluation with, for example, hematoxylin-eosin, helps to ensure that small changes are not missed and that background and artifactual changes, which may include vacuoles, pigments, and dark neurons, are not over-interpreted as compound-related changes. The antigen-antibody reaction appears simple and clear in theory, but many steps might go wrong. Remember that methods including the antigen-antibody complex rely on handling/fixation of tissues or cells, antibody shipping/storing issues, antibody titration, temperature/duration of antibody incubation, visualization of the antibody and interpretation of the results. Optimize staining protocols to the material you are using.