Establishing and experimental protocol for making transparent brain for neurodegenerative disease research

Abstract

Until recently, different techniques have been developed to clear tissues to become transparent. However, to speed up the process, most of the methods involve many steps of chemical treatments or extreme temperature. This leads to several issues including fluorescent quenching, protein degradation, and distortion to tissue architecture due to swelling/shrinkage. While some techniques protect tissues by cross-linking them with acrylamide and epoxy monomers, these treatments are usually irreversible. Until now, there is no technique that can preserve tissues from exposing to many steps of chemicals or extreme temperature. Therefore, we have established a delipidation-based technique to preserves tissue structure without the use of any external monomer network. In this study, we performed partial delipidation by adding OPTIClear, a refractive index (RI) matching solution, into SDS solution. We found that tissue clearing with SDS-OPTIClear not only reduced the required clearing time than usual SDS solution, but also showed less protein loss and no intense swelling/shrinkage. We further demonstrated that tissues cleared with SDS-OPTIClear would have better antigen preservation, thus allowing better quality in immunohistochemical staining. Previously, it has been reported the failure of immunofluorescence staining with Iba-1, a marker of microglia, after SDS-treatment. Here, we demonstrated Iba-1 immunoreactivity in the hippocampus treated with SDS-OPTIClear and SDS solution. Under the same condition, tissues cleared with SDS-OPTIClear showed better morphology and deeper penetration depth compared to the one cleared with SDS. Immunohistochemical staining of other antigens such as ChAT and TOMM-20 showed that they had stronger signal and deeper penetration depth in tissues treated with SDS-OPTIClear. Finally, we performed TO-PRO-3 staining on tissues and visualized under confocal microscopy before and after clearing such that the degree of distortion after clearing can be examined and quantified. While tissues cleared with SDS showed dramatic size change upon clearing and RI matching, SDS-OPTIClear did not trigger swelling effect. Tissue morphology was preserved throughout clearing and RI matching. Taken together, we demonstrated that SDS-OPTIClear preserved better tissue morphology and architecture without the requirement of tissue embedding. This is a simple and cost-effective methodology. This allows us to investigate neurodegeneration with high resolution in a three-dimensional manner without intense swelling/shrinkage.