Double labeling with fluorescence in situ hybridization in Drosophila whole-mount embryos.

Previously, we described a technique for fluorescence in situ hybridization (FISH) in whole-mount Drosophila embryos using fluoresceinlabeled RNA probes (2). This technique provides several advantages over conventional alkaline phosphatasebased detection methods. For example, a fluorescent label is nondiffusible and so allows higher-resolution imaging. Furthermore, it allows the use of confocal and deconvolution microscopy, which can take advantage of this enhanced resolution and at the same time penetrate deeply into the sample to give three-dimensional information. Another potential advantage of fluorescence-based detection methods is the possibility to generate double-labeled samples in which relative overlaps between two or more expression patterns can be resolved at the cellular and subcellular levels. Our previous method allowed for the detection of only one transcript. Here we describe modifications of our protocol, which allow simultaneous detection of two different transcripts. We also describe a variation that combines whole-mount FISH with fluorescent antibody staining. Double FISH was achieved using a combination of fluoresceinand digoxigenin-tagged RNA probes. The probes were detected by incubating first with anti-fluorescein and anti-digoxigenin antibodies and subsequently with the corresponding non-cross-reacting fluorescently tagged secondary antibodies. Several probes were tested with equally successful results. Strong signals were detected using conventional fluorescence microscopy. RNA probes were made using T7 or T3 RNA polymerase (both from Boehringer Mannheim, Laval, QC, Canada) essentially as described in the manufacturer’s specification sheet. Enzyme buffers (10×) provided with the polymerases were used. Flourescein-12UTP and digoxigenin-12-UTP ribonucleotides were obtained as premixed 10× cocktails containing appropriate concentrations of unlabeled ribonucleotides (Fluorescein RNA Labeling Mix and DIG RNA Labeling Mix; Boehringer Mannheim). Before use, template DNA must be cut to completion and carefully extracted with phenol/chloroform and then chloroform alone, followed by ethanol precipitation. RNase-free reagents are recommended but are not essential if care is used in preparation of the template and assembly of the transcription reactions. To eliminate possible RNase contamination, 1 μL RNase inhibitor (RNAguard; Pharmacia Biotech, Baie dÌUrfe, QC, Canada) (1 U/μL) was added to each 25-μL transcription reaction. Following the transcription reaction (at 37°C for 2 h), the labeled probes were precipitated by the addition of 1 μL 0.5 M EDTA, 2.5 μL 4 M LiCl and 75 μL absolute ethanol. The resulting pellet was washed with 500 μL cold 70% ethanol and resuspended in 100 μL RNase-free H2O. Figure 1 shows the products of two transcription reactions resolved on a 0.8% agarose gel and stained with ethidium bromide. Degradation of the probe by carbonate buffer treatment (Reference 3, Protocol 6) was found to be not only unnecessary but usually detrimental.