X-gal staining of Drosophila embryos compatible with antibody staining or in situ hybridization.

the slow development. The low background especially applies for detection of digoxigenin-labeled RNA probes. When detecting identical probes labeled with fluorescein, the background is higher, presumably because of nonspecific binding of the fluorescein antibody (the higher background is detected with both BCIP and NBT/BCIP). In cases in which the investigated signals are of different intensity, we obtained the best results when the probe giving the strongest signal was labeled with fluorescein. Another consideration is the expression domains of the investigated genes. For overlapping expression domains, the blue color produced with BCIP will be masked by the purple precipitate produced with NBT/BCIP. Thus, in cases in which the expression domains of the investigated genes overlap, the blue color must be designated to the largest expression domain (Figure 1C). Another option is to photograph the specimen before the secondary color reaction is carried out. We have used double labeling in situ hybridization to investigate gene interactions in mouse limb buds (Figure 1, C, D and F). However, there are no obvious reasons why this method could not be applied to other organisms or protocols.