Comparative hybridization protocols in zebrafish.

hybridization is a commonly used technique in molecular biology to assess the temporal and spatial expression of a given gene. As a long and labor-intensive protocol, double hybridization, which detects two genes in series, is challenging and can require a lot of troubleshooting. Optional additives, polyvinyl alcohol and dextran sulfate, were tested in a standard hybridization protocol and several colorimetric stain pairings using double hybridization in zebrafish embryos. Optional additives can improve staining time and reduce nonspecific background. Nitro-blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate (BCIP) + Fast Red/BCIP was the most effective stain pairing. As a proof-of-concept, this work shows that and are expressed in distinct regions of the developing zebrafish brain.