Single and Double FISH Protocols for Drosophila

In situ hybridization within whole-mount Drosophila tissues was made routine with the introduction of digoxigenin labeled probes and alkaline phosphatase based detection methods (1 ). However, this method of detection until recently has been limited by the required use of alkaline phosphatase conjugated secondary antibodies and chromogenic substrates. The use of alkaline phosphatase substrates and their diffusible products limits the resolution of staining, particularly in thick tissues and deep within the embryo. Without additional probes, double-labeling and interpretation of results is also very difficult.