Optimization of the optical transparency of bones by PACT-based passive tissue clearing.

Recent developments in tissue clearing methods such as the passive clearing technique (PACT) have allowed three-dimensional analysis of biological structures in whole, intact tissues, thereby providing a greater understanding of spatial relationships and biological circuits. Nonetheless, the issues that remain in maintaining structural integrity and preventing tissue expansion/shrinkage with rapid clearing still inhibit the wide application of these techniques in hard bone tissues, such as femurs and tibias. Here, we present an optimized PACT-based bone-clearing method, Bone-mPACT+, that protects biological structures. Bone-mPACT+ and four different decalcifying procedures were tested for their ability to improve bone tissue clearing efficiency without sacrificing optical transparency; they rendered nearly all types of bone tissues transparent. Both mouse and rat bones were nearly transparent after the clearing process. We also present a further modification, the Bone-mPACT+ Advance protocol, which is specifically optimized for processing the largest and hardest rat bones for easy clearing and imaging using established tissue clearing methods.

Investigation of PRDM10 and PRDM13 Expression in Developing Mouse Embryos by an Optimized PACT-Based Embryo Clearing Method.

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.