In Situ Hybridization to Localize mRNAs

In situ hybridization (ISH) to localize sites of expression of mRNA is widely applicable to studies of invasion and metastasis in human pathology specimens and tissues from experimental animals or cell cultures. ISH can provide crucial information about where a specific gene is expressed, before suitable antisera can be made. The ICRF In Situ Hybridization Service has hybridized more than 44,000 sections of principally formalin-fixed, paraffin wax-embedded tissues, with a high success rate. Our preferred method for screening for mRNAs in routinely fixed paraffin wax-embedded materials uses 35S-labeled riboprobes, and has been developed over several years from the method described by Senior and colleagues (1). It may seem unfashionable to use an isotopic method, but (b35)S provides the best balance of specificity, sensitivity, and cost; most importantly, unlike nonisotopic ISH, it gives reproducible and easily interpretable results without the need to adjust the method to individual cell types within sections. For alternative methodology for fluorescent in situ hybridization (FISH) techniques, please refer to Chapter 14 by Goker and Shipley. The protocol described here gives good results for the vast majority of routine surgical specimens, and useful data from about half of the blocks sampled to date from the discarded archive of a General Hospital Pathology Department (avoiding postmortem specimens). With the economies of scale, reagent costs are approximately US $11 (UK £6.50) per slide. Comparison of different published protocols for ISH shows that steps regarded as essential by one group may not be used by another group. Nevertheless, there are some common principles and checkpoints, and only one needs to be wrong for the entire experiment to fail.