Simple Rescue of Opaque Tissue Previously Cleared by iDISCO

Recent advancements in tissue-clearing techniques and volumetric imaging have greatly facilitated visualization and quantification of biomolecules, organelles, and cells in intact organs or even entire organisms. Generally, there are two types of clearing methods: hydrophobic and hydrophilic (i.e., clearing with organic or aqueous solvents, respectively). The popular iDISCO approach and its modifications are hydrophobic methods that involve dehydration, delipidation, decolorization (optional), decalcification (optional), and refractive-index (RI) matching steps. Cleared samples are often stored for a relatively long period of time and imaged repeatedly. However, cleared tissues can become opaque over time, which prevents accurate reimaging. We reasoned that the resurgent haziness is likely due to rehydration, residual lipids, and uneven RI deep inside those tissue samples. For rescue, we have developed a simple procedure based on iDISCO. Beginning with a methanol dehydration, samples are delipidated using dichloromethane, followed by RI matching with dibenzyl ether (DBE). This simple method effectively re-clears mouse brains that have turned opaque during months of storage, allowing the user to effectively image immunolabeled samples over longer periods of time. Key features • This simple protocol rescues previously cleared tissue that has turned opaque. • The method does not cause detectable loss of immunofluorescence from previously stained samples. Graphical overview.