Fast free-of-acrylamide clearing tissue (FACT)-an optimized new protocol for rapid, high-resolution imaging of three-dimensional brain tissue.

Fast Free-of-Acrylamide Clearing Tissue (FACT) is a new sodium dodecyl sulfate (SDS)-based clearing protocol for the chemical clearing and imaging of brain tissue containing transgenic or immunolabeled fluorescent proteins. In the present study, we have developed this new method and optimized multiple dimensions of the workflow, including reduced clearing time, improved efficiency of fluorescent signals without the need for electrophoretic or complex instrumentations, preservation of cytoarchitectural details, optimized confocal microscopy, and accelerated data collection. We systematically compared seven clearing protocols with the FACT protocol, using transgenic mouse brains with fluorochrome expression in microglia. Only six days were required for detecting transgene-labeled markers in a 1-mm thick brain slice from adult mice, and 14 days were required for detecting antibody-labeled markers in the same-sized tissue. Preservation of fluorescent signal was achieved by decreasing clearing time, adjusting the pH of the SDS solution, and using the appropriate temperature for tissue clearing, all of which contributed to the superiority of our method. We conclude that the FACT protocol can be successfully applied to the fluorescent imaging of mouse brain tissue, and will facilitate structural analyses and connectomics of large assemblies of cells and their networks in the context of three-dimensional organ systems.