Radma Mahmood and Ivor Mason 1 Introduction

The development of such techniques as transgenesis, saturation mutagenesis, and the polymerase chain reaction (PCR), all of which are described in detail elsewhere in this volume, has revolutionized experimental embryology. However, no single procedure has been more broadly applied across the field than that of in situ hybridization to RNA. This allowed researchers to determine rapidly the spatial and temporal expression of their gene of interest without having to resort to more tedious and less certain approaches requiring the production of antisera against its protein product. As such, in situ hybridization has become the standard first step toward the characterization of the developmental significance of a newly identified gene. Of course, in situ hybridization tells you nothing regarding translation of the mRNA into protein or about protein's subsequent localization, modification, or stability. However, in situ hybridization remains an extremely powerful tool in enabling the researcher to predict likely developmental functions for gene products rapidly.

Historically, this technique was pioneered by the Angerers working with sea urchin embryos (1). It was refined by several groups, notably by McMahon and Wilkinson in studies of gene expression in sections of mouse embryos (2,3). During the past five years or so, the use of radioactive in situ hybridization of tissue sections has been largely superseded by the application of non-radioactive approaches on whole embryos at early developmental stages and, to a lesser extent, on sections of older embryos and adults. The latter approaches offer considerable advantages in terms of their speed and, in the case of in situ hybridisations to whole embryos, further advantages in the ease of spatial interpretation of results. However, the radioactive approach remains more sensitive and is the only method

From: Methods in Molecular Biology, Vol. 97: Molecular Embryology: Methods and Protocols Edited by: P. T. Sharpe and I. Mason © Humana Press Inc., Totowa, NJ

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Fig. 1. Adjacent sagittal sections of an E14.5 mouse embryo. (A) Bright-field view of toluidine blue-stained section. (B) Same field viewed under dark ground optics to show hybridization of FGF-7 probe to a number of tissues, including a subset of muscles, nasal epithelium, cartilage capsules, and lung. Section was exposed for 14 d (C). Adjacent section hybridized with a cardiac a-actin probe showing hybridization to all skeletal and cardiac muscle. Section was exposed for 24 h.

by which certain "low-abundance" transcripts are detectable. Furthermore, for these reasons, it is likely that many studies of developmental gene expression undertaken by nonradioactive, whole embryo approaches are incomplete. As such, radioactive in situ hybridization remains a valid approach in embryological and other fields. The following protocol (4) is that used in our laboratory (see Fig. 1) and is a modification of an original method of Wilkinson and Green (5).

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