The crucial step is the inactivation and removal of the first applied Fab-AP conjugate using the low pH step after the first staining round. Inactivation of the first applied Fab-AP conjugate by heating is not recommended, since incubation at 65C for 20 min or at 80C for 10 min causes fading of the red precipitate resulting in a weaker and more diffuse signal. In contrast, the blue precipitate darkens resulting in an increased background in the embryo and a nearly black yolk. Because the second round of detection is less sensitive than the first, stain the red first. Nevertheless, if necessary, it is also possible to do the purple staining first.
Fluorescein labeled probes are slightly less sensitive than digoxigenin probes.
Therefore the stronger probe is usually detected in red using a fluorescein probe. Antibodies, Stock Solutions and Reagents Preadsorption of antibody with zebrafish embryos Zebrafish embryos need not to be dechorionated. The number of embryos you use for preabsorption depends on the amount of antibody you want to preabsorb. The stages used for preabsorption should include the same or older stages you will use in your in situ hybridizations. 2-3 d embryos work well.