In Situ Hybridization of Zebrafish Embryos

Embryos were fixed in 4% paraformaldehyde at 4°C overnight and stored in methanol at -20°C. 24-hpf embryos were permeabilized in 2% hydrogen peroxide in methanol for 20 minutes (min) at room temperature (RT) and stepwise rehydrated to PBST (phosphate buffered saline, 0.1% Tween-20 pH 7.3). After rehydration, embryos were further permeabilized by a 10-min proteinase K treatment and postfixed for 20 min with 4% paraformaldehyde at RT. Prehybridization, probe hybridization and washes were performed as described [5 (link)] except for addition of 5% dextran sulfate to the hybridization buffer. In two-color experiments, digoxigenin- and dinitrophenol-labeled probes were mixed together in hybridization buffer at the appropriate concentrations and hybridized simultaneously.