Radio-Isotopic In Situ Hybridization on Tissue Sections: Practical Aspects and Quantification

in situ hybridization has become a powerful tool for the analysis of gene expression within a topographical context and has become indispensable in developmental studies. Different strategies are to be used for different purposes. Whole mount in situ hybridization allows the rapid global analysis of changes in the spatiotemporal patterns of gene expression. Its use is limited to early developmental stages owing to penetration problems with increasing size of the embryo. Analysis of the precise location of the expressing cells requires subsequent sectioning of the stained embryo. Provided its sensitivity would be high enough, nonradioactive in situ hybridization would be the preferred choice owing to its superior resolving power. With the possible exception of early embryonic stages, it is our experience that the sensitivity of nonradioactive in situ hybridization on sections is inferior to that of the radio-isotopic procedure. Moreover, the sensitivity see ms probe-dependent and the signal is difficult to quantify.