The overall goal of Chromogenic Multi-target WISH is to facilitate the mapping of mRNA distribution patterns within intact Drosophila embryos.With this method, the mRNA localisations of three different genes can be directly compared within the same embryo.Multi-color WISH can be used to generate gene expression maps in developing organisms in a fast and reliable way.This can provide important clues on gene regulatory interactions, active and embryonic development, or organogenesis.The main advantage of this technique is that mRNA expression patterns of different genes are visualized in distinctive and contrasting colors.This way, unique and overlapping expression sites can be highlighted with high accuracy.Using standard procedures, collect, dechorionate, fix, and devitellinize the required embryos.Store them in methanol in the freezer until they're needed.Now, begin with the embryos at room temperature.Fit an insert into the first well of a 12 well plate and add two milliliters of methanol.Transfer the desired amount of embryos into the insert which is used to carry the embryos throughout the procedure.Now, rehydrate the embryos using three minute incubations in a decreasing methanol series.Start with 75%methanol in PBS.Follow with 50%methanol in PBS-T.Then, use 25%methanol in PBS-T and finish with two incubations in pure PBS-T.Next, prefix the embryos in 4%paraformaldehyde in PBS for 20 minutes at room temperature.Remove the fixative using four washes in PBS-T.During the wash steps, thaw and prepare the working solutions for Proteinase K and glycine.After the washes, permeabilize the embryos in freshly prepared Proteinase K for three minutes at room temperature.Stop the reaction using two one minute washes in the freshly prepared glycine.Then, postfix the embryos in 4%paraformaldehyde made in PBS for 20 minutes at room temperature.Wash out the paraformaldehyde with four washes in PBS-T three minutes a piece.Then, transfer the embryos to prehybridization buffer.They may be stored in the freezer until needed.Begin with distributing the embryos in prehy buffer into two milliliter tubes.Aliquot about 15 microliters of settled embryos into each tube.Now, incubate the embryos in a 65
