Investigating urea is an ideal additive to the Accu-OptiClearing delipidation cocktail

The brain is a complicated tissue with high lipid content. Different tissue clearing methods have been used to study its complex structures and functions. However, the balance between myelin preservation and clearing efficiency remains an obstacle in brain tissue clearing. In the previous study, Accu-OptiClearing has been developed to overcome this challenge by combining detergent and RI matching solution in the delipidation solution. This successfully preserves brain tissue structure and lipid content, but the clearing efficiency can be further improved. Here, we investigated the effects of adding urea as a non-detergent permeabilizing agent to the delipidation solution of Accu-OptiClearing by measuring the clearing efficiency, protein loss, tissue size change and myelin preservation. Different concentrations of urea (2 M, 4 M, 6 M and 8 M) were added to the 10% HxD-OPTIClear solution for comparison. From the results, we found that the 2 M urea additive can achieve sufficient improvement in clearing efficiency with unchanged tissue preservation and probe compatibility. These findings demonstrate that the addition of non-detergent urea could potentially modify the Accu-OptiClearing delipidation solution. Hence, it opens room for screening other non-detergent chemicals in optimizing the Accu-OptiClearing delipidation cocktail for future neuroscience studies.