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Published November 2015 | Supplemental Material + Accepted Version
Journal Article Open

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


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.

Additional Information

© 2015 Macmillan Publishers Limited. Published online 22 October 2015. We thank H. McBride, D.K. Newman and J. Allman for useful discussions on uses of tissue clearing across disciplines. We thank M. Brissova and A.C. Powers from Vanderbilt University for providing fixed human pancreas tissue used in Figure 1 and guidance with pancreatic markers and anatomy. This work was funded by grants to V.G.: the US National Institutes of Health (NIH) Director's New Innovator IDP20D017782-01; the NIH/National Institute on Aging (NIA) 1R01AG047664-01; the Beckman Institute for Optogenetics and CLARITY; the Pew Charitable Trust; and the Kimmel Foundation. Work in the Gradinaru Laboratory at Caltech is also funded by awards from the following (to V.G.): the NIH Brain Research through Advancing Innovative Neurotechnologies (BRAIN) 1U01NS090577; the NIH/National Institutes of Mental Health (NIMH) 1R21MH103824-01; the Human Frontiers in Science Program; the Mallinckrodt Foundation; the Gordon and Betty Moore Foundation through grant GBMF2809 to the Caltech Programmable Molecular Technology Initiative; the Michael J. Fox Foundation; Caltech-GIST; and the Caltech-City of Hope Biomedical Initiative. This work was also supported by grants to P.J.B. from the NIH (2 P50 GM082545-06; W.I. Sundquist, principal investigator) and gifts from the Gordon and Betty Moore Foundation and the Agouron Institute to support electron microscopy at Caltech; and by National Science Foundation (NSF) IIS-1253538 and DBI-1262547 grants to C.C.F. K.Y.C. and N.C.F. were supported by the NIH Predoctoral Training in Biology and Chemistry (2T32GM007616-36). Contributions: J.B.T. and V.G. wrote the manuscript with input from all coauthors. J.B.T., K.Y.C., N.C.F., B.Y., B.E.D. and V.G. designed and performed experiments, analyzed the data and prepared figures. C.C.F. wrote the data analysis section, including associated figures and data analysis; C.X. assisted with tissue clearing and imaging for data sets in this section. A.G., A.L., L.C. and V.G. planned for and built the light sheet and collected and analyzed the associated data. M.S.L., P.J.B. and V.G. planned and performed TEM tissue processing and imaging and prepared the EM figure. V.G. supervised all aspects of the project. All authors edited and approved the manuscript. The authors declare no competing financial interests.

Attached Files

Accepted Version - nihms-785590.pdf

Supplemental Material - nprot.2015.122-S1.pdf

Supplemental Material - nprot.2015.122-S2.zip

Supplemental Material - nprot.2015.122-S3.zip

Supplemental Material - nprot.2015.122-S4.zip

Supplemental Material - nprot.2015.122-SF1.jpg

Supplemental Material - nprot.2015.122-SF2.jpg

Supplemental Material - nprot.2015.122-SF3.jpg

Supplemental Material - nprot.2015.122-SF4.jpg

Supplemental Material - nprot.2015.122-SF5.jpg

Supplemental Material - nprot.2015.122-SF6.jpg

Supplemental Material - nprot.2015.122-SF7.jpg


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Additional details

August 20, 2023
October 24, 2023