CLARITY is a novel tissue clearing technique that transforms intact biological tissues into a nanoporous hydrogel-tissue hybrid, preserving anatomical structures, proteins and nucleic acids. The hydrogel-based structure is transparent after the removal of lipids and permits several rounds of immunostaining and imaging. This technique provides an ideal way for researchers to examine the central nervous system (i.e., mouse brain and spinal cord) intact. CLARITY was selected as one of ten breakthroughs in 2013 by Science. However, the original CLARITY technique still has severe technical limitations which impede its application in wider fields. Therefore, many modified clearing methods based on CLARITY have emerged. As all CLARITY-based tissue clearing techniques involve similar procedures, the present review attempted to divide these methods into individual procedures in order to provide new ways to test and combine tissue clearing methods. Furthermore, the combination of clearing methods could help to determine the optimal method for clearing and imaging large samples.

CLARITY is a hydrogel embedding clearing method that has the advantages of transparency, different tissue compatibility and immunostaining compatibility. However, there are also some limitations to CLARITY as it requires a long time to achieve transparency, and the electrophoresis clearing is complex. Therefore, we aimed to simplify the electrophoresis system and shorten the processing time of CLARITY. In our study, we developed a non-circulation electrophoresis system to achieve easier manipulation of electrophoresis clearing. We modified the original CLARITY protocol in hydrogel embedding methods, clearing buffer and immunostaining. When comparing brains processed by our modified method or the original protocol, we found our modifications permit faster and more efficient clearing and labeling. Moreover, we developed a new clearing method named Passive pRe-Electrophroresis CLARITY (PRE-CLARITY) and a new immunostaining method named Centrifugation-Expansion staining (CEx staining). PRE-CLARITY achieved faster clearing and higher transparency, and CEx staining accomplished intact mouse brain labeling faster. With our modifications to CLARITY, we accomplished intact mouse brain clearing and immunostaining within one week, while this requires weeks to months with the original CLARITY. Our studies would allow high-content tracing and analysis of intact brain or other large-scale samples in a short time.