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- In all steps, take care not to mix heavily or allow air interface contacts with the embryos. When the protocol calls for "washing" embryos, add 1000 ul of solution to side of the eppendorf tube. This results in gentle mixing of the embryos with the wash solution. Don't squirt solutions directly onto the embryos. If needed the tubes can be given a mild extra mix with a gentle inversion to make sure that the solution is equally distributed among the embryos and that they are not stuck to each other. Since you are trying to keep them intact over 3 days of washing, its important to be gentlygentle.
- Reading up on a Northern protocol is a good point of reference to better understand the purpose of all the steps in the protocol. The in situ and the Northern protocols mirror each other because both use RNA anti-sense probes to target RNA.
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