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