Ethyl Cinnamate-Based Tissue Clearing Strategies

Tissue clearing turns otherwise turbid and opaque tissue transparent, enabling imaging deep within tissues. The nontransparent nature of most tissues is due to the refractive index mismatch between its three major constituent components (lipids, proteins, and water). All tissue clearing methods rectify this mismatch by homogenizing the refractive index within the tissue and carefully matching it to the surrounding media. Here we describe a detailed protocol to clear a wide range of salamander tissues. We also include several optional steps such as depigmentation, antibody staining, and tissue mounting. These steps are optional, and do not change anything in the steps needed for tissue clearing. Depending on the fluorescent signal and optics employed, images up to several millimeters inside of the tissue can be acquired.