Two-color detection of mRNA transcript localizations in fish and fly embryos using alkaline phosphatase and beta-galactosidase conjugated antibodies

A fast and simplified two-color in situ hybridization procedure for the sim ultaneous detection of two different mRNAs in whole-mounted zebrafish and D rosophila embryos is presented. Transcript distributions are detected in a single incubation step using a mixture of alkaline phosphatase and beta-gal actosidase coupled antibodies. The different transcripts are visualized in contrasting colors by the use of beta-galactosidase substrates that develop color precipitates (magenta, blue) easily distinguishable from those of th e standard alkaline phosphatase substrates. This protocol can be followed b y standard immunohistochemistry to detect the expression of a third gene (a t the protein level) in a third color.