A multiwell plate approach to increase the sample throughput during tissue clearing

Tissue clearing, coupled with immunostaining, enables the transition from two-dimensional to three-dimensional pathology and has the potential to substantially improve data quality for biomedical diagnostics. Nevertheless, the workflows are limited by the complex sample processing protocols. Approaches for the parallel processing of samples, to include tissue clearing, immunostaining, imaging and analysis can increase three-dimensional pathology throughput. Here we detail a step-by-step approach that combines a tissue clearing device with a six-well multiwell plate to increase the throughput compared with methods using conventional clearing protocols. The six-well multiplate allows for parallel tissue clearing of multiple samples and is compatible with passive tissue clearing methods including Clear, Unobstructed Brain/Body Imaging Cocktails and Computational (CUBIC) analysis. In addition, gel embedding is performed without moving the samples from the wells, and a series of steps such as imaging with a high-speed light-sheet microscope and analysis in the cloud can be performed. Although this procedure slightly extends the overall time required for preparing and analyzing a single sample, it reduces the effort required at each step, such as reagent exchange and gel embedding, which results in an overall reduction in hands-on time due to the parallel sample processing. We describe a series of whole-organ analyses, including high-throughput tissue clearing, staining, gel embedding, imaging and data analysis in the cloud, as a useful platform for cellular biology and pathology. The total process varies depending on the presence or absence of immunostaining, but for some six-well plates, the tissue clearing process, imaging and data analysis can be completed within 10 d.