Abstract
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.
Highlights
Microglia are the resident parenchymal myeloid cells of the central nervous system (CNS)
Our systematic comparisons of Free-of-Acrylamide Clearing Tissue (FACT) with seven previously established clearing techniques demonstrate that rapid clearing times as well as preservation of cellular proteins and fine cytoarchitectural details are all achievable with FACT in 1-mm brain slices
The other currently available methods, such as CLARITY11, PACT13, and SWITCH19, are all hydrogel-based, and it has been shown that cellular proteins can be trapped in polyacrylamide hydrogel-embedded tissue by linkage to hydrogel molecules[11]
Summary
Microglia are the resident parenchymal myeloid cells of the central nervous system (CNS). New and improved methods are urgently needed for the simultaneous evaluation of large populations of cells such as microglia in three dimensions, with a focus on fine details of their cytoarchitecture and their structural contacts with surrounding cells[5] Because of their higher expression levels, transgenic fluorescent proteins have stronger and more visible signals than antibody-stained markers and require shorter tissue preparation times. Developed for the large-scale imaging of transparent and intact tissues with an emphasis on brain neural circuits, including BABB6, Scale7, 3DISCO8, ClearT9, SeeDB10, CLARITY11, passive CLARITY12, PACT13, CUBIC14, 15, and FASTClear[16] Of these approaches, the ones that clear tissue by replacing the water in the tissue with organic solvents, such as BABB and 3DISCO, cannot prevent the quenching of fluorescent protein signals for longer than two days[6, 8, 10]. For long-term imaging, the deformation of tissues caused by hydrogel expansion during clearing limits the usefulness of these powerful methods for evaluating fine structures such as microglia branches and neuronal processes
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