See-Star: a versatile hydrogel-based protocol for clearing large, opaque and calcified marine invertebrates.

Studies of morphology and developmental patterning in adult stages of many invertebrates are hindered by opaque structures, such as shells, skeletal elements, and pigment granules that block or refract light and necessitate sectioning for observation of internal features. An inherent challenge in studies relying on surgical approaches is that cutting tissue is semi-destructive, and delicate structures, such as axonal processes within neural networks, are computationally challenging to reconstruct once disrupted. To address this problem, we developed See-Star, a hydrogel-based tissue clearing protocol to render the bodies of opaque and calcified invertebrates optically transparent while preserving their anatomy in an unperturbed state, facilitating molecular labeling and observation of intact organ systems. The resulting protocol can clear large (> 1 cm³) specimens to enable deep-tissue imaging, and is compatible with molecular techniques, such as immunohistochemistry and in situ hybridization to visualize protein and mRNA localization. To test the utility of this method, we performed a whole-mount imaging study of intact nervous systems in juvenile echinoderms and molluscs and demonstrate that See-Star allows for comparative studies to be extended far into development, facilitating insights into the anatomy of juveniles and adults that are usually not amenable to whole-mount imaging.