Background

Tissue clearing paired with light-sheet microscopy rejuvenated the century-old histochemistry field [1]. Clearing can render organs or even whole animals transparent [2], while the light-sheet microscopy can image fluorescent labels deep inside tissues or bodies [3]. Available clearing techniques generally fall into two categories based on the solvents used, specifically whether using hydrophilic or hydrophobic (including hydrogel-based methods) solutions [4]. Despite technical differences, the underlying principles between the two are the same, i.e., substituting molecules that diffract lights (e.g., lipids, pigments, and calcium phosphate) with other molecules that match the refractive index (RI) of the imaging media [2]. The transparency of cleared tissues allows fluorescence imaging of biomolecules deep inside the tissue (i.e., intrinsically expressed fluorescent proteins or after labeling with fluorescently tagged antibodies), which is further facilitated by light-sheet microscopy that illuminates the sample using an adjustable thin sheet of UV light [3]. Notably, fluorophore-tagged antibodies are often used to boost the detection of endogenous fluorescent proteins. iDISCO, a hydrophobic clearing method integrated with immunohistochemistry [5], has become very popular and given rise to multiple derivatives including uDISCO [6] and vDISCO [7]. Conventionally, cleared samples are stored in RI-matching media, often for months, for repeated imaging (please see https://idisco.info for more information). However, during storage, iDISCO-cleared specimens can turn cloudy and some even become opaque, which makes them unsuitable for imaging accurately by light-sheet microscopy. We suspected that such changes in tissue opacity with storage arose from residual lipids, a loss of RI matching deep inside the tissue, or the rehydration of samples by moisture in the air or dissolved in the storage solvent. To counteract those changes, we developed a simple rescue protocol based on immunofluorescence-compatible iDISCO. The approach is comprised by three steps—dehydration, delipidation, and RI rematching. The protocol we describe can successfully re-clear entire, murky mouse brains that had been previously cleared by iDISCO. We imaged the re-cleared brains by light-sheet microscopy, observing little or no loss of immunofluorescence as compared to imaging prior to storage, allowing the samples to be stored and studied far beyond the time of initial scanning.