Nov 22, 2019

Public workspaceWhole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping

DOI
dx.doi.org/10.17504/protocols.io.8xehxje
  • Jennifer B Treweek1,
  • Ken Y Chan1,
  • Nicholas C Flytzanis1,
  • Bin Yang1,
  • Benjamin E Deverman2,
  • Alon Greenbaum1,
  • Antti Lignell3,
  • Cheng Xiao1,
  • Long Cai3,
  • Mark S Ladinsky1,
  • Pamela J Bjorkman1,
  • Charless C Fowlkes4,
  • Gradinaru Lab1
  • 1Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California, USA;
  • 2Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California, USA.;
  • 3Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California, USA;
  • 4Department of Computer Science, University of California, Irvine, California, USA
  • Optical Clearing of Tissue
  • Neurodegeneration Method Development Community
Gradinaru Lab
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DOI: dx.doi.org/10.17504/protocols.io.8xehxje
External link: https://www.nature.com/articles/nprot.2015.122
Protocol Citation: Jennifer B Treweek, Ken Y Chan, Nicholas C Flytzanis, Bin Yang, Benjamin E Deverman, Alon Greenbaum, Antti Lignell, Cheng Xiao, Long Cai, Mark S Ladinsky, Pamela J Bjorkman, Charless C Fowlkes, Gradinaru Lab 2019. Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping. protocols.io https://dx.doi.org/10.17504/protocols.io.8xehxje
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 01, 2019
Last Modified: November 22, 2019
Protocol Integer ID: 29382
Abstract

To facilitate fine-scale phenotyping of whole specimens, we describe here a set of tissue fixation-embedding, detergent-clearing and staining protocols that can be used to transform excised organs and whole organisms into optically transparent samples within 1–2 weeks without compromising their cellular architecture or endogenous fluorescence. PACT (passive CLARITY technique) and PARS (perfusion-assisted agent release in situ) use tissue-hydrogel hybrids to stabilize tissue biomolecules during selective lipid extraction, resulting in enhanced clearing efficiency and sample integrity. Furthermore, the macromolecule permeability of PACT- and PARS-processed tissue hybrids supports the diffusion of immunolabels throughout intact tissue, whereas RIMS (refractive index matching solution) grants high-resolution imaging at depth by further reducing light scattering in cleared and uncleared samples alike. These methods are adaptable to difficult-to-image tissues, such as bone (PACT-deCAL), and to magnified single-cell visualization (ePACT). Together, these protocols and solutions enable phenotyping of subcellular components and tracing cellular connectivity in intact biological networks.

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