303 — Aqueous-based tissue clearing in crustaceans

Aqueous tissue clearing has been gaining a lot of traction recently. But one of the most common tissue-clearing methods — CUBIC — doesn’t work well on crustaceans. That’s because crustaceans have calcified exoskeletons with very deep pigmentation: Even though the skeleton isn’t too thick, it’s very tough to see through, which anyone who has ever tried to hold a lobster up to the light no doubt knows.

In order to make CUBIC work on these animals, it was necessary to develop a pretreatment process. In this process, Konno & Okazaki first decalcified the exoskeleton with EDTA in order to allow the bleaching agent to fully bathe the tissue; bleaching was then performed with H₂O₂.

This process — which didn’t work as well on insect exoskeletons — likely indicates that the majority of pigmentation in the tissue of isopods is fixed in the calcified exoskeleton. (Clearing mammalian or other vertebrate tissue doesn’t suffer from this same restriction, since vertebrate pigmentation tends to be relatively diffuse.) But it also provides an avenue to cleared-tissue study in crustaceans, a family of common model organisms for a variety of biological processes.