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Nat Protoc. 2015 Nov;10(11):1860-1896. doi: 10.1038/nprot.2015.122. Epub 2015 Oct 22.

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

Author information

1
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California, USA.
2
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California, USA.
3
Department of Computer Science, University of California, Irvine, California, USA.
#
Contributed equally

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.

PMID:
26492141
PMCID:
PMC4917295
DOI:
10.1038/nprot.2015.122
[Indexed for MEDLINE]
Free PMC Article

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