Modified CLARITY Achieving Faster and Better Intact Mouse Brain Clearing and Immunostaining

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.