Effects of lipofuscin on in situ hybridization in human neuronal tissue

In situ hybridization is a highly sensitive technique for detecting nucleic acid sequences within tissues, and is frequently employed in neurovirology. However, this technique requires many appropriate controls in order to recognize and avoid potential artifactual hybridization. We have encountered abundant reaction to lipofuscin in neurons in human peripheral and central nervous systems, using various DNA probes, which could be misinterpreted as positive signals. This pseudohybridization reaction was resistant to treatment with RNase or DNase and was also present in tissue sections treated with hybridization mixture or nuclear autoradiographic emulsion in the absence of any radioactive probes. Characteristics used to distinguish between authentic in situ hybridization and the reaction to neuronal lipofuscin include cellular localization, color, margins and granular appearance, sensitivity to treatment with nucleases and the effect of exposure time on signal intensity. These guidelines should be used to avoid potential misinterpretation of in situ hybridization results with human tissue.