ClearT: a detergent- and solvent-free clearing method for neuronal and non-neuronal tissue.

Department of Pathology and Cell Biology, Columbia University, College of Physicians and Surgeons, 630 West 168th Street, 14-509 P&S, New York, NY 10032, USA.
Development (Impact Factor: 6.27). 03/2013; 140(6):1364-8. DOI: 10.1242/dev.091844
Source: PubMed

ABSTRACT We describe a clearing method for enhanced visualization of cell morphology and connections in neuronal and non-neuronal tissue. Using Clear(T) or Clear(T2), which are composed of formamide or formamide/polyethylene glycol, respectively, embryos, whole mounts and thick brain sections can be rapidly cleared with minimal volume changes. Unlike other available clearing techniques, these methods do not use detergents or solvents, and thus preserve lipophilic dyes, fluorescent tracers and immunohistochemical labeling, as well as fluorescent-protein labeling.

1 Bookmark
  • Science 01/2015; 347(6221):474-5. DOI:10.1126/science.aaa5084 · 31.48 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Optogenetic constructs have revolutionized modern neuroscience, but the ability to accurately and efficiently assess their expression in the brain and associate it with prior functional measures remains a challenge. High-resolution imaging of thick, fixed brain sections would make such post-hoc assessment and association possible; however, thick sections often display autofluorescence that limits their compatibility with fluorescence microscopy. We describe and evaluate a method we call “Brain BLAQ” (Block Lipids and Aldehyde Quench) to rapidly reduce autofluorescence in thick brain sections, enabling efficient axon-level imaging of neurons and their processes in conventional tissue preparations using standard epifluorescence microscopy. Following viral-mediated transduction of optogenetic constructs and fluorescent proteins in mouse cortical pyramidal and dopaminergic neurons, we used BLAQ to assess innervation patterns in the striatum, a region in which autofluorescence often obscures the imaging of fine neural processes. After BLAQ treatment of 250–350 μm-thick brain sections, axons and puncta of labeled afferents were visible throughout the striatum using a standard epifluorescence stereomicroscope. BLAQ histochemistry confirmed that motor cortex (M1) projections preferentially innervated the matrix component of lateral striatum, whereas medial prefrontal cortex projections terminated largely in dorsal striosomes and distinct nucleus accumbens subregions. Ventral tegmental area dopaminergic projections terminated in a similarly heterogeneous pattern within nucleus accumbens and ventral striatum. Using a minimal number of easily manipulated and visualized sections, and microscopes available in most neuroscience laboratories, BLAQ enables simple, high-resolution assessment of virally transduced optogenetic construct expression, and post-hoc association of this expression with molecular markers, physiology and behavior.
    Frontiers in Neuroanatomy 02/2015; 9(6). DOI:10.3389/fnana.2015.00006 · 4.18 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Visualizing the three-dimensional morphology and spatial patterning of cells embedded deep within dense connective tissues of the musculoskeletal system has been possible only by utilizing destructive techniques. Here we utilize fructose-based clearing solutions to image cell connectivity and deep tissue-scale patterning in situ by standard confocal microscopy. Optical clearing takes advantage of refractive index matching of tissue and the embedding medium to visualize light transmission through a broad range of bovine and whole mount murine tissues, including cartilage, bone, and ligament, of the head and hindlimb. Using non-destructive methods, we show for the first time intercellular chondrocyte connections throughout the bulk of cartilage, and we reveal in situ patterns of osteocyte processes and the lacunar-canalicular system deep within mineralized cortical bone. Optical clearing of connective tissues is expected to find broad application for the study of cell responses in normal physiology and disease pathology.
    PLoS ONE 01/2015; 10(1):e0116662. DOI:10.1371/journal.pone.0116662 · 3.53 Impact Factor


Available from
May 27, 2014