CUBIC-plus: An optimized method for rapid tissue clearing and decolorization

Advanced CUBIC clearing method can clear diverse organs and even the entire body of a mouse to enable high-resolution 3D imaging. Advanced CUBIC reagent has often been used due to its low toxicity and easy preparation. However, Advanced CUBIC has a long experimental cycle, which reduces the efficiency of data acquisition. In this study, we first tracked the clarity changes of different organs cleared by Advanced CUBIC and identified the shortest time required for optimal transparency in individual organs. We then introduced ultrasound processing and developed a decolorization cocktail to optimize the clearing efficiency of the Advanced CUBIC method. With the optimized clearing CUBIC-Plus method, we achieved high resolution 3D imaging of mouse organs. Our CUBIC-Plus provides an efficient procedure to clear different organ for 3D imaging at high resolution.