In Situ Hybridization Protocols

Abstract

2nd ed, edited by Ian A. Darby (Methods in Molecular Biology, vol 123), 343 pp, with illus, Totowa, NJ, Humana Press, 2000.The second edition of In Situ Hybridization Protocols has a new editor and is substantially different in its stated aims and intended audience. While the first edition tried to cover as many areas of the in situ hybridization (ISH) technique as possible, the goals of the second are to describe research methods for gene mapping and localization of messenger RNA expression in tissues. This is a slimmer volume than the first edition, with more emphasis on tissue techniques than on chromosomal techniques. There are 2 clinical application chapters at the end, compared with 5 in the first edition.New chapters include those on ISH to whole mount embryo specimens, ISH at the electron microscope level, and detection of DNA fragmentation in apoptosis. However, a fair amount of the material in the new edition is repeated from the 1994 edition without substantial change. For example, both chapters on ISH to polytene chromosomes are nearly the same as in the first edition, down to the illustrations and references, and both cover similar material. Another topic included in both editions is radioactive ISH for gene mapping. (Even in the early 1980s when this was the only technique available, many researchers found it entirely too tedious. It is hard to imagine that anyone would try to master this time-consuming procedure rather than pursue other alternatives.)When learning a new technique, figures demonstrating equipment or results, comparative tables, notes, and background information can be especially helpful to novices. As one would expect from a multiauthored anthology, the chapters vary considerably in this regard. The new chapter on ISH with electron microscopy has beautiful pictures of several applications, as well as detailed protocols, discussion, and notes. The chapter on localization of polymerase chain reaction–amplified DNA is copiously illustrated with examples of what can go wrong and contains extensive explanatory notes. However, the chapter on fiber fluorescence ISH doesn't contain a single figure, not even of a successful result.As the editor notes in the preface, ISH techniques span the fields of molecular biology and histology/cytology, making them difficult to master. In fact, most of the techniques described in this volume are not at all simple, requiring expertise not only in molecular biology and histology, but also in tissue culture, cytogenetics, electron microscopy, statistics, or other areas. Le Moine, the author of the chapter on in situ hybridization using radioactive probes to study gene expression, acknowledges that ”the entire procedure is quite complicated” (page 152). I know from personal experience how hard it was to get the radioactive ISH technique to work, starting with a good background in cytogenetics and autoradiography and a mediocre knowledge of molecular techniques. So I do have to wonder if it might not be easier and even cheaper, at least for your first foray into these complex protocols, to just go on a field trip to learn from an expert. If you're an intermediate looking to expand your ISH repertoire, you should be able to pick up some tips from this book.