The following method was developed specifically for identifying transcripts with overlapping or colocalized expression domains (37). It is therefore useful for identifying ectopically expressed transgene products in transient transgene experiments along with upregulated transcripts of downstream genes. Double in situ hybridizations are performed by hybridization with a mixture of fluorescein- and digoxigenin-labeled antisense RNA probes followed by sequential incubation in AP-conjugated antibodies to fluorescein and digoxigenin, respectively. The first antibody is stained with Fast Red (see Note 10) and the second with enzyme labeled fluorescence (ELF™) substrate. The signals are viewed on a fluorescence microscope with rhodamine and DAPI filter sets, respectively.
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