Abstract
This chapter reviews protocols and instructions for designing appropriate controls and tips for trouble-shooting different problems. It should be borne in mind that most of these techniques are still under development because improvements are made continuously. For in situ hybridization with two probes, one probe is transcribed exactly as described previously, and the second probe is transcribed by the same method except that FITC-labeled uridine triphosphate (UTP; Boehringer-Mannheim) mixed with unlabeled nucleotides replaces the digoxigenin-nucleotide mix (the proportion of FITC-labeled to unlabeled nucleotides is described in the sheet supplied by the manufacturers). In experiments, it is often desirable to study the distribution of different mRNAs and proteins at a given stage of development in one embryo. For example, it may be of interest to detect the ectopic expression of a gene introduced with a retroviral vector, at the same time as studying the expression of one or more of its putative target genes.
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