The DeepClear protocol was first described by Pende et al. 2020. Sci. Adv. 6, eaba0365. [doi:10.1126/sciadv.aba0365] For leech embryonic materials, the result of this protocol is similar to CUBIC. Its clearing performance on the adult leech is better than CUBIC. However, meglumine diatrizoate, an ingredient of DeepClear Solution 2, is quite expensive. For large specimens, the cost could be prohibitive. As an alternative, we found using CUBIC Solution 2 in place of DeepClear Solution 2 yielded similar results (at least for adult leech specimens).

Clearing protocol:

1. Following the fixation of the leech or the leech embryos (typically in 4% formaldehyde in 0.5X PBS at 4°C overnight), wash the specimens in PBS several times.

2. Incubate the specimens in pre-chilled acetone at -20°C for 30 minutes.

3. Wash the specimens with PBS for three times.

4. Incubate the specimen in 0.5X Solution 1 at 37°C, with rotation or gentle shaking. Then, incubate the specimen in 1X solution 1 at 37°C, with rotation or gentle shaking, overnight.

5. Wash the specimens with PBS for three times.

6. Incubate and mount the specimens in solution 2.

• Solution 1: 10% (v/v) THEED [2,2′,2″,2″′-(Ethylenedinitrilo)tetraethanol], 5% (v/v) Triton X-100, 25% Urea (w/v)

• Solution 2: 50% meglumine diatrizoate (w/w) in PBS (pH9-9.3)