MAX: a simple, affordable, and rapid tissue clearing reagent for 3D imaging of wide variety of biological specimens.

Transparency of biological specimens is crucial to obtaining detailed 3-dimensional images and understanding the structure and function of biological specimens. This transparency or tissue clearing can be achieved by adjusting the refractive index (RI) with embedding media and removing light barriers such as lipids, inorganic deposits, and pigments. Many currently available protocols consist of multiple steps to achieve sufficient transparency, making the process complex and time-consuming. Thus, in this study, we tailored the recipe for RI adjustment media named MAX based on the recently reported MACS protocol to achieve a single-step procedure, especially for ECM-rich tissues. This was achieved by the improvement of the tissue penetrability of the RI-matching reagent by combining MXDA with sucrose or iodixanol. While this was sufficient for the 3D imaging in many applications, MAX can also be combined with modular processes for de-lipidation, de-coloration, and de-calcification to further maximize the transparency depending on the special features of the tissues. Our approach provides an easy alternative for tissue clearing and 3D imaging.