We describe a strategy for developing hydrophilic chemical cocktails for tissue delipidation, decoloring, refractive index (RI) matching, and decalcification, based on comprehensive chemical profiling. More than 1,600 chemicals were screened by a high-throughput evaluation system for each chemical process. The chemical profiling revealed important chemical factors: salt-free amine with high octanol/water partition-coefficient (logP) for delipidation, N-alkylimidazole for decoloring, aromatic amide for RI matching, and protonation of phosphate ion for decalcification. The strategic integration of optimal chemical cocktails provided a series of CUBIC (clear, unobstructed brain/body imaging cocktails and computational analysis) protocols, which efficiently clear mouse organs, mouse body including bone, and even large primate and human tissues. The updated CUBIC protocols are scalable and reproducible, and they enable three-dimensional imaging of the mammalian body and large primate and human tissues. This strategy represents a future paradigm for the rational design of hydrophilic clearing cocktails that can be used for large tissues.

Whole-organ/body three-dimensional (3D) staining and imaging have been enduring challenges in histology. By dissecting the complex physicochemical environment of the staining system, we developed a highly optimized 3D staining imaging pipeline based on CUBIC. Based on our precise characterization of biological tissues as an electrolyte gel, we experimentally evaluated broad 3D staining conditions by using an artificial tissue-mimicking material. The combination of optimized conditions allows a bottom-up design of a superior 3D staining protocol that can uniformly label whole adult mouse brains, an adult marmoset brain hemisphere, an ~1 cm3 tissue block of a postmortem adult human cerebellum, and an entire infant marmoset body with dozens of antibodies and cell-impermeant nuclear stains. The whole-organ 3D images collected by light-sheet microscopy are used for computational analyses and whole-organ comparison analysis between species. This pipeline, named CUBIC-HistoVIsion, thus offers advanced opportunities for organ- and organism-scale histological analysis of multicellular systems.

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The localization of human vitamin D receptor (VDR) in the absence of its ligand 1,25-dihydroxyvitamin D 3 was investigated using chimera proteins fused to green fluorescent protein (GFP) at either the N or C terminus, and the nuclear localization signal (NLS) was identified. Plasmids carrying the fusion proteins were transiently or stably introduced into COS7 cells, and the subcellular distribution of the fusion proteins was examined. GFPtagged wild-type VDRs were located predominantly in nuclei but with a significant cytoplasmic presence, while GFP alone was equally distributed throughout the cells. 10 ؊8 M 1,25-dihydroxyvitamin D 3 promoted the nuclear import of VDR in a few hours. To identify the NLS, we constructed several mutated VDRs fused to GFP. Mutant VDRs that did not bind to DNA were also localized predominantly in nuclei, while the deletion of the hinge region resulted in the loss of preference for nucleus. A short segment of 20 amino acids in the hinge region enabled cytoplasmic GFP-tagged alkaline phosphatase to translocate to nuclei. These results indicate that 1) VDR is located predominantly in nuclei with a significant presence in cytoplasm without the ligand and 2) an NLS consisting of 20 amino acids in the hinge region facilitates the transfer of VDR to the nucleus. The vitamin D receptor (VDR)1 is one of the ligand-dependent transcription factors that make up the nuclear hormone receptor superfamily (1-3). To modulate the transcription of target genes in response to its cognate ligand 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ), VDR must be localized in nucleus and then bind to an enhancer designated as the vitamin D-responsive element (VDRE), forming a heterodimer with retinoid X receptor (1-6). In contrast to the case for the glucocorticoid receptor (GR), which translocates from the cytoplasm to the nucleus when exposed to its ligand, VDR does not bind to heat shock protein 90, and both immunocytochemical and biochemical fractionation studies suggested the nuclear localization of VDR even in the absence of 1,25(OH) 2 D 3 (7-10).Several reports, however, demonstrated that VDR was located in cytoplasm in the absence of ligand and transported to nucleus in response to 1,25(OH) 2 D 3 (11-13). Although the reason for conflicting results as to the distribution of VDR is not clear, the fixation and cell permeabilization procedures in immunostaining might influence the subcellular distribution of the subject protein. Consistent with this explanation, Barsony et al. (11), by the fixation of cells using a microwave, revealed the cytoplasmic localization of VDR in contrast to the nuclear localization detected by a conventional fixation method utilizing the same antibody against VDR.To avoid the fixation and cell permeabilization steps required in the immunostaining procedure, in the present study we have taken advantage of fusion with green fluorescent protein (GFP), which has been proven to be a useful tag for monitoring the subcellular distribution and trafficking of various proteins in living cell...

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