iDISCO: A Simple, Rapid Method to Immunolabel Large Tissue Samples for Volume Imaging

Abstract

The visualization of molecularly labeled structures within large intact tissues in three dimensions is an area of intense focus. We describe a simple, rapid, and inexpensive method, iDISCO, that permits whole-mount immunolabeling with volume imaging of large cleared samples ranging from perinatal mouse embryos to adult organs, such as brains or kidneys. iDISCO is modeled on classical histology techniques, facilitating translation of section staining assays to intact tissues, as evidenced by compatibility with 28 antibodies to both endogenous antigens and transgenic reporters like GFP. When applied to degenerating neurons, iDISCO revealed unexpected variability in number of apoptotic neurons within individual sensory ganglia despite tight control of total number in all ganglia. It also permitted imaging of single degenerating axons in adult brain and the first visualization of cleaved Caspase-3 in degenerating embryonic sensory axons in vivo, even single axons. iDISCO enables facile volume imaging of immunolabeled structures in complex tissues. PAPERCLIP:

Use of labeled tomato lectin for imaging vasculature structures

“…The extensive labeling of vasculature by intravenously applied tomato lectin also opens the possibility of revealing extensive and detailed three-dimensional images of the vasculature of any organ through use of new techniques of tissue clearance (Ertürk et al 2012;Chung et al 2013;Renier et al 2014). …”

Stochastic electrotransport selectively enhances the transport of highly electromobile molecules

“…For example, many emerging tissue clearing techniques use surfactant micelles to directly remove lipids from a tissue and thus eliminate light-scattering boundaries to improve optical penetration for holistic visualization (2,(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25), but transporting these micelles into the intact tissue via diffusion can take weeks (2,15). Although electrophoresis can speed up this process, as demonstrated in CLARITY, its application has been limited to low electric fields because using high fields can damage tissue structures (2).…”

“…However, with the development of in situ molecular interrogation methods (6,13,14) and tissue clearing techniques (2,(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25) and an emphasis on studying organ-scale tissue as a whole, a pressing need has arisen for a means of expediting the transportation of various molecules into intact tissues. For example, many emerging tissue clearing techniques use surfactant micelles to directly remove lipids from a tissue and thus eliminate light-scattering boundaries to improve optical penetration for holistic visualization (2,(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25), but transporting these micelles into the intact tissue via diffusion can take weeks (2,15).…”

“…However, these techniques have been mostly confined to small samples owing to the difficulty of labeling and examining deep structures in large-scale intact tissues (6,(27)(28)(29)(30)(31). CLARITY and other emerging tissue-clearing techniques (2,(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25) render intact tissues optically transparent and macromolecule-permeable, allowing examination deep inside Significance Many chemical and biomedical techniques rely on slow diffusive transport because existing pressure-based methods or electrokinetic methods can incidentally damage the sample. This study introduces a novel transport concept termed stochastic electrotransport that can selectively and nondestructively expedite transport of electromobile molecules into a porous sample, such as fixed biological tissues.…”

Novel imaging tools for investigating the role of immune signalling in the brain

“…The physical properties of the lipid bilayer prevents large macromolecules for example, antibodies, from readily diffusing deep into the tissue whilst the molecular heterogeneity of the lipids themselves alter the refractive index of the tissue, further perturbing the excitation and emission of light from fluorescently-labelled probes (Richardson & Lichtman 2015). To overcome these problems, "clearing" methods such as CLARITY, PARS, CUBIC, SeeDB, ScalA2, iDISCO and 3DISCO have been developed (Susaki et al 2015;Bin Yang et al 2014;Renier et al 2014;Tomer et al 2014;Susaki et al 2014;Ke et al 2013;Becker et al 2012;Hama et al 2011). …”