iDISCO

iDISCO stands for immunolabeling-enabled three-dimensional imaging of solvent-cleared organs. It is a tissue clearing technique that combines immunolabeling of intact biological specimens with solvent-based clearing to render tissues optically transparent and compatible with 3D fluorescence imaging. The method enables whole-organ labeling and imaging, allowing researchers to visualize cellular and molecular architecture within intact samples, such as the mouse brain, spinal cord, or other organs.

The iDISCO workflow typically begins with fixation of tissue, followed by permeabilization and immunostaining, using antibodies to tag proteins of interest. After labeling, samples undergo a graded solvent dehydration and clearing sequence, commonly involving organic solvents that extract lipids and then replace the tissue with a refractive-index-matching solvent. The cleared samples are then imaged with light-sheet fluorescence microscopy or other 3D imaging modalities. The resulting data permit three-dimensional mapping of neural circuits, vascular networks, immune cell distributions, and tumor cells within whole organs.

A variant, iDISCO+, introduced to improve antibody penetration and labeling reliability in larger samples, often including methanol pretreatment and optimized antibody incubation conditions. The iDISCO family has been widely adopted for developmental biology and neuroscience, with additional derivatives such as uDISCO addressing whole-body clearing.

Limitations include potential tissue distortion from solvent exchange, loss or quenching of certain fluorophores, and the need for specialized high-containment facilities and imaging equipment. Despite these considerations, iDISCO and its successors have become foundational tools for 3D anatomical mapping and connectomics in vertebrate and invertebrate specimens.