The CUBIC protocol was first reported by Susaki et al., 2014. Cell 157, 726-739. [doi:10.1016/j.cell.2014.03.042]. Its clearing effect on the yolky early leech embryos is by far the best (in terms of cost-performance ratio) among the plethora of aqueous clearing protocols that we have tested. It also works on adult transgenic leeches.

1. Clearing protocol:

2. Incubate fixed specimens in a 1:1 mix of Solution 1 and water at 37ºC for 1 hour (optional: add nuclear counterstain).

3. Incubate fixed specimens in Solution 1 at 37ºC for 1 hour, repeat twice more, followed by an overnight incubation (3X 1-hour incubation + 1X O/N
   incubation; nuclear counterstain can be added in all steps).

4. Wash the specimens with PBS (with 0.01% Sodium Azide) six times, 10 minutes each.

5. Incubate the specimens in Solution 2 for 1 hour, and repeat three times. 

• Solution 1: 25%(w/w) Urea, 25%(w/w) Quadrol [N,N,N′,N′-Tetrakis(2-Hydroxypropyl)ethylenediamine], 15% (w/w) Triton X-100, 35% (w/w) H2O 

• Solution 2: 25% (w/w) Urea, 50%(w/w) Sucrose, 10%(w/w) Triethanolamine, 15% (w/w) H2O