Detection of mRNA by Whole Mount in situ Hybridization and DNA Extraction for Genotyping of Zebrafish Embryos.

ABSTRACT

In situ hybridization is used to visualize the spatial distribution of gene transcripts in tissues and in embryos, providing important information about disease and development. Current methods involve the use of complementary riboprobes incorporating non-radioactive labels that can be detected by immunohistochemistry and coupled to chromogenic or fluorescent visualization. Although recent fluorescent methods have allowed new capabilities such as single-molecule counting, qualitative chromogenic detection remains important for many applications because of its relative simplicity, low cost and high throughput, and ease of imaging using transmitted light microscopy. A remaining challenge is combining high contrast signals with reliable genotyping after hybridization. Dextran sulfate is commonly added to the hybridization buffer to shorten development times and improve contrast, but this reagent inhibits PCR-based genotyping. This paper describes a modified protocol for in situ hybridization in fixed whole mount zebrafish embryos using digoxigenin (DIG) labeled riboprobes that are detected with alkaline phosphatase conjugated anti-DIG antibodies and nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl-phosphate (BCIP) chromogenic substrates. To yield embryos compatible with downstream genotyping after hybridization without sacrificing contrast of the signal, this protocol omits dextran sulfate and utilizes a lower hybridization temperature.