CUBIC-f: An optimized clearing method for cell tracing and evaluation of neurite density in the salamander brain

Abstract

BACKGROUND: Although tissue clearing and subsequent whole-brain imaging is now possible, standard protocols need to be adjusted to the innate properties of each specific tissue for optimal results. This work modifies exiting protocols to clear fragile brain samples and documents a downstream pipeline for image processing and data analysis. NEW METHOD: We developed a clearing protocol, CUBIC-f, which we optimized for fragile samples, such as the salamander brain. We modified hydrophilic and aqueous' tissue-clearing methods based on Advanced CUBIC by incorporating Omnipaque 350 for refractive index matching. RESULTS: By combining CUBIC-f, light sheet microscopy and bioinformatic pipelines, we quantified neuronal cell density, traced genetically marked fluorescent cells over long distance, and performed high resolution characterization of neural progenitor cells in the salamander brain. We also found that CUBIC-f is suitable for conserving tissue integrity in embryonic mouse brains. COMPARISON WITH EXITING METHODS: CUBIC-f shortens clearing and staining times, and requires less reagent use than Advanced CUBIC and Advanced CLARITY. CONCLUSION: CUBIC-f is suitable for conserving tissue integrity in embryonic mouse brains, larval and adult salamander brains which display considerable deformation using traditional CUBIC and CLARITY protocols.