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Recent developments in tissue clearing methods such as CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/In situ hybridization-compatible Tissue hYdrogel) have allowed for the three-dimensional analysis of biological structures in whole, intact tissue, providing greater understanding of spatial relationships and biological circuits. Nonetheless, studies have reported issues with maintaining structural integrity and preventing tissue disintegration, preventing the wide application of these techniques to fragile tissues such as developing embryos. Here, we present optimized passive clearing techniques, mPACT-A, that improve tissue rigidity without the expense of optical transparency. We also present a further modified mPACT-A protocol that is specifically optimized for handling mouse embryos, which are small and fragile, such that they easily dismantle when processed via established tissue clearing methods. We demonstrate proof-of-concept by investigating the expression of two relatively understudied PRDM proteins, PRDM7 and PRDM12, in intact cleared mouse embryos at various stages of development. We observed strong PRDM7 and PRDM12 expression in the developing mouse nervous system, suggestive of potential roles in neural development that will be tested in future functional studies., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest in this work., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Recent developments in tissue clearing methods have significantly advanced the three-dimensional analysis of biological structures in whole, intact tissue, providing a greater understanding of spatial relationships and biological circuits. Nonetheless, studies have reported issues with maintaining structural integrity and preventing tissue disintegration, limiting the wide application of these techniques to fragile tissues such as developing embryos. Here, we present an optimized passive tissue clearing technique (PACT)-based embryo clearing method, initial embedding PACT (IMPACT)-Basic, that improves tissue rigidity without compromising optical transparency. We also present IMPACT-Advance, which is specifically optimized for thin slices of mouse embryos past E13.5. We demonstrate proof-of-concept by investigating the expression of two relatively understudied PR domain (PRDM) proteins, PRDM10 and PRDM13, in intact cleared mouse embryos at various stages of development. We observed strong PRDM10 and PRDM13 expression in the developing nervous system and skeletal cartilage, suggesting a functional role for these proteins in these tissues throughout embryogenesis. [ABSTRACT FROM AUTHOR]