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

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.

StereoMate: 3D Stereological Automated Analysis of Biological Structures

“…The first step was to develop an immunofluorescence labelling protocol suitable for thick or wholemount tissue specimens ( Figure 1a). A key problem with previous tissue clearing methodologies is their impact on antibody affinity to their antigens 17,18 , and so the first goal was to develop a methodology that removed lipid membrane barriers to enhance antibody penetration into tissue, yet still maintained tissue antigenicity.…”

HSP70/IL-2 Treated NK Cells Effectively Cross the Blood Brain Barrier and Target Tumor Cells in a Rat Model of Induced Glioblastoma Multiforme (GBM)

“…We utilized fast Free-of-Acrylamide Clearing Tissue (FACT), which has been recently established [55], as a third protocol to study NK cells in vivo. Herein, we employed this tissue clearing method to achieve fluorescent three-dimensional imaging of rat brain tissue.…”

Application of Airy beam Light sheet microscopy to examine early neurodevelopmental structures in 3D hiPSC-derived human cortical spheroids

“…These aqueous clearing techniques reduce light scattering by removing tissue lipids and replacing them with a clear matrix, and as reported by Xu et al (2017) can also facilitate greater penetration of antibodies. The combined effect enables significant preservation of both tissue ultrastructure and the accessibility of native proteins to antibody probes [22,24,25]. To date only a limited number of studies have applied the use of LSM or tissue clearing in combination with human brain organoids.…”

“…Several methods are now available to render intact tissues optically transparent [22][23][24][25]. These aqueous clearing techniques reduce light scattering by removing tissue lipids and replacing them with a clear matrix, and as reported by Xu et al (2017) can also facilitate greater penetration of antibodies.…”

“…Several methods are now available to render intact tissues optically transparent [22][23][24][25]. These aqueous clearing techniques reduce light scattering by removing tissue lipids and replacing them with a clear matrix, and as reported by Xu et al (2017) can also facilitate greater penetration of antibodies. The combined effect enables significant preservation of both tissue ultrastructure and the accessibility of native proteins to antibody probes [22,24,25].…”