In situ hybridization: Introduction to techniques, applications and pitfalls in the performance and interpretation of assays

In situ hybridization (ISH) has become a common laboratory technique used for the analysis of gene expression and for the localization of specific DNA and RNA molecules in cells. Many different methods of performing ISH have been described. These techniques have evolved into important tools in basic scientific research and in clinical diagnoses. One of the goals of ISH is to localize gene sequences in situ and to visualize the products within cells while preserving cell integrity. This allows for meaningful anatomical and histological interpretation of the localized product(s) within heterogeneous tissues. Because of the possibility of false positive and false negative results that may occur with ISH assays, familiarity with the pathophysiology of the molecules that are analyzed and the cellular processes involved as well as with limitations of the assays can help to avoid erroneous diagnoses with clinical specimens.