Abstract

In situ proximity ligation assay (isPLA) is an increasingly popular technique that aims at detecting the close proximity of two molecules in fixed samples using two primary antibodies. The maximal distance between the antibodies required for producing a signal is 40 nm, which is lower than optical resolution and approaches the macromolecular scale. Therefore, isPLA may provide refined positional information, and is commonly used as supporting evidence for direct or indirect protein-protein interaction. However, we show here that this method is inherently prone to false interpretations, yielding positive and seemingly specific signals even for totally unrelated antigens. We discuss the difficulty to produce adequate specificity controls. We conclude that isPLA data should be considered with extreme caution.