Fundamental principles of in situ hybridization

In situ hybridization provides invaluable information regarding the localization of gene expression in heterogeneous tissues. The technique is extremely sensitive and can detect the amount of mRNA contained in a single cell. This review provides a starting point for those who wish to begin using in situ hybridization in their own laboratories. The procedure outlined here is based on 35S-labeled riboprobes and has been used with many probes and tissues with a greater than 90% success rate on the first hybridization. The importance of appropriate controls is stressed. Clusters of silver grains after hybridization do not necessarily indicate specific mRNA localization. Regions of the tissue rich in nuclei often appear to cause spurious binding of probes and have high backgrounds often mistaken as positive signals. The most difficult aspect of in situ hybridization is not to get clusters of silver grains on the slide but rather to do the appropriate controlled experiments to ensure that the signal is real and is not due to some artifactual binding of the probe to the tissue.