Application of alkaline phosphatase-mediated azo dye staining for dual fluorescent in situ hybridization in zebrafish

Abstract

We report a dual fluorescent in situ hybridization (FISH) method for direct comparison of cellular d istributions of different gene transcripts in the embryonic zebrafish brain and other tissues. After simultaneous hybridization of two differently labeled antisense RNA probes, the different hapten labels are visualized by peroxidase (POD)-mediated deposition of fluorochrome-labeled tyramides and alkaline phosphatase- based Fast Blue or Fast Red chromogenic staining, respectively. Since chromogenic Fast Blue and Fast Red precipitates display red-fluorescent emission, multiplexed visualization of different transcripts is possible by combination with carboxyfluorescein-labeled tyramides, which show emission in the green spectrum. The application of differential reporter enzymes provides advantages over procedures using sequential POD detection. The POD-coupled and AP-coupled antibodies can be mixed together in a single incubation step reducing the required time, spent otherwise for extensive washings and multiple incubations. In addition, removal or inactivation of antibody-POD conjugates as required in sequential POD detection procedures can be omitted. Therefore, potential false-positive detection of co-localization by insufficient inactivation is prevented.