Procedures

1. After antibody wash, remove PBS + detergent from the embryos.
2. Mix BM Purple in the bottle by shaking.
3. Add ~100 microliters of BM Purple to embryos (<50 embryos)
4. Allow color development in dark.
   1. - it may take overnight or more if in room temperature.
   2. - it is OK to develop color in 37 °C; check every hour if so.
   3. - it is OK to alternate between room temperature and 37 °C over the course of several days if it is necessary.
5. Wash with PBS + 0.1% Tween 20 several times.
6. Inspect under a dissection microscope to see if the crystal precipitation is fully removed.

Note:

Use BM Purple for in situ hybridization of low-abundance or rare transcript; in situ hybridization to abundant transcripts and immunostaining tend to produce unusable over-staining, if not carefully monitored.