Single-Cell Phenotyping within Transparent Intact Tissue through Whole-Body Clearing
Understanding the structure-function relationships at cellular, circuit, and organ-wide scale requires 3D anatomical and phenotypical maps, currently unavailable for many organs across species. At the root of this knowledge gap is the absence of a method that enables whole-organ imaging. Herein, we present techniques for tissue clearing in which whole organs and bodies are rendered macromolecule-permeable and optically transparent, thereby exposing their cellular structure with intact connectivity. We describe PACT (passive clarity technique), a protocol for passive tissue clearing and immunostaining of intact organs; RIMS (refractive index matching solution), a mounting media for imaging thick tissue; and PARS (perfusion-assisted agent release in situ), a method for whole-body clearing and immunolabeling. We show that in rodents PACT, RIMS, and PARS are compatible with endogenous-fluorescence, immunohistochemistry, RNA single-molecule FISH, long-term storage, and microscopy with cellular and subcellular resolution. These methods are applicable for high-resolution, high-content mapping and phenotyping of normal and pathological elements within intact organs and bodies.
© 2014 Elsevier Inc. Received: May 6, 2014; Received in revised form: July 13, 2014; Accepted: July 15, 2014; Published Online: July 31, 2014. We thank the entire Gradinaru lab for helpful discussions. We also thank Drs. Dianne Newman and David Anderson for helpful discussions and suggestions. We thank Drs. Amir Arbabi and Andrei Faraon for assistance with SEM imaging. We thank Richard Bennett and Erik Cabral for assistance with tumor procurement. We thank Leica Microsystems for acquiring some of the images presented with a Leica TCS SP8 with Leica HC FLUOTAR L 25×/1.00 IMM CORR objective designed for CLARITY imaging. B.D. and V.G. especially wish to acknowledge the contributions of Paul H. Patterson (1943–2014), whose desire to apply tissue clearing to unresolved questions in neuroscience pushed us to further evolve these methods. This work was funded by: the NIH/NINDS New Innovator (NIH IDP20D017782-01); NIH 1R01AG047664-01; Startup funds from the President and Provost of California Institute of Technology and the Biology and Biological Engineering Division of California Institute of Technology; the Beckman Institute of Caltech; the Pew Charitable Trust; the Sidney Kimmel Foundation (to V.G.). And NIH R01HD075605 (to L.C.). V.G. is also supported by: Human Frontiers in Science Program, the Mallinckrodt Foundation, the Gordon and Betty Moore Foundation, the Michael J. Fox Foundation, Caltech-GIST, NIH 1R01NS085910-01, NIH 1R21MH103824-01. C-K.C., S.S., E.L., acknowledge support from the Caltech Biology Division Training grant (NIH/NRSA 5T32GM07616). R.P.K. acknowledges support from NIH/NIAMS (5T32AR058921). J.B.T. acknowledges the Colvin Postdoctoral Fellowship and CALTECH Division of BBE. B.Y. and V.G. have disclosed these methods and findings to the Caltech Office of Technology Transfer (CIT File No.: CIT-6686-P). V.G. is a cofounder of Circuit Therapeutics and holds related IP and stock (less than 5%). At this writing, V.G. receives no research funding, royalties, or consultant fees from any for-profit organization. Author Contribution: B.Y., J.B.T., and V.G. conceived the project. B.Y., J.B.T., R.P.K., B.E.D., C.-K.C., E.L., S.S., L.C., and V.G. planned and executed experiments. B.Y., J.B.T., V.G. made the figures and wrote the paper with input from all other authors. V.G. supervised all aspects of the work.
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