Methods and probes for cleared tissue: an imperfect table

Feb 25, 2016
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TF table methagora post

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In the March issue of Nature Methods, the technology feature explores some ways that labs are optimizing probes to image cleared tissue. As we interviewed scientists, we learned about published work and ongoing unpublished experiences. Here is a snapshot of how some probes work with some clearing methods. It’s an imperfect table and is likely to evolve as research continues. We welcome your comments. We know there are different viewpoints and varying experiences and we hope it will be helpful to others to hear about them.

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Probe Labeling shown in large samplesb Labeling shown in small samples Does not work well?
Genetically encoded fluorescent proteins 3DISCO (signal quenched after a few days), CLARITY, CUBIC, ExM (variant)a, PACT/PARS (signal retained 6 months and longer)a, sDISCOa BABB (signal quenched after a few hours), ClearT2, ExM (variant)a , ExM/ePACTa , ScaleA2, ScaleS, SeeDB,
Sucrose, TDE
Immunolabels 3DISCO, CLARITY, iDISCO, iDISCO+a, iSeeDB, PACT/PARS, Sucrose, SWITCH BABB, ClearT, ClearT2, CUBIC, SeeDB, SeeDB2a (in press) Sucrose, TDE, ExM
Specific nucleic acid detection EDC-CLARITY CLARITY, ExM (variant)a, PACT/PARS
Dyes and stains
Congo Red 3DISCO
Lipophilic dyes such as DiI, Sudan Black PACT/PARS (Sudan Black), SWITCH (DiI) ClearT, ScaleS, SeeDB
Nuclear stains such as DAPI, DRAQ5, SYTO and PI CUBIC, ExM (variant)a, PACT/PARS, SWITCH SeeDB, SeeDB2a
SNAP-tags with SiR probes PACT/PARS, BABB, Scale SeeDB

Clearing method: Solvent-based; simple immersion; hyperhydration; hydrogel-based.

aUnpublished information

bIndicates larger samples such as whole organs

Sources: E. Boyden, MIT; K. Chung, MIT, K. Deisseroth, Stanford University; H-U. Dodt, Vienna University of Technology/Medical University of Vienna; V. Gradinaru, Caltech; P. Heppenstall, EMBL; Takeshi Imai, RIKEN; ­­K. Johnsson, EPFL; J. Lichtman, D. Richardson, Harvard University; A. Miyawaki, RIKEN; M. Tessier-Lavigne, N. Renier, Rockefeller University.

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Glossary of some tissue clearing agent acronyms

3DISCO : Three-dimensional imaging of solvent-cleared organs

sDISCO: Stabilized three-dimensional imaging of solvent-cleared organs

BABB : Benzyl alcohol and benzyl-benzoate

CLARITY : Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/In situ hybridization-compatible Tissue-hYdrogel

CUBIC : Clear unobstructed brain imaging cocktails and computational analysis

EDC-CLARITY : 1-Ethyl-3-3-dimethyl-aminopropyl carbodiimide-CLARITY

ePACT: PACT-based expansion clearing.

iDISCO : Immunolabeling-enabled 3D imaging of solvent-cleared organs

iDISCO+ : Immunolabeling-enabled 3D imaging of solvent-cleared organs plus

PACT : Passive Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/In situ hybridization-compatible Tissue-hYdrogel

PARS : Perfusion-assisted agent release in situ

SeeDB : See Deep Brain

Spalteholz’s preparation : Benzylbanzoate/methylsalicate

TDE : 2,2'-thiodiethanol

Some lab resource pages

Chung lab resources – Literature, protocols, videos and discussion pages from the MIT lab of Kwanghun Chung related to SWITCH, electrostochastic transport and CLARITY.

CLARITY Resources – Protocols, literature, data and videos related to CLARITY, developed in the lab of Karl Deisseroth at Stanford University. Links to CLARITY Wiki and CLARITY Forum

Expansion microscopy resources – Literature and protocols related to expansion microscopy developed in the MIT lab of Edward Boyden.

iDISCO resources – Literature, protocols and information about validated antibodies related to iDISCO

SeeDB Resources – Protocols, literature videos related to SeeDB developed in the RIKEN lab of Takeshi Imai.

Natalie de Souza

Chief Editor, Nature Methods

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