Department of Neurosurgery, The Spine and Spinal Cord Institute, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul 06273, Korea.
Brain Korea 21 PLUS Project for Medical Science, Yonsei University, Seoul 03722, Korea.
Int J Mol Sci. 2021 Mar 12;22(6):2892. doi: 10.3390/ijms22062892.
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.
近年来，组织透明化方法的发展显著促进了整个完整组织中生物结构的三维分析，使我们更深入地了解空间关系和生物回路。然而，研究报告指出，这些技术在维持结构完整性和防止组织解体方面存在问题，限制了这些技术在脆弱组织（如发育中的胚胎）中的广泛应用。在这里，我们提出了一种优化的基于被动组织透明化技术（PACT）的胚胎透明化方法，初始包埋 PACT（IMPACT）-Basic，该方法在不影响光学透明度的情况下提高了组织的刚性。我们还提出了专门针对 E13.5 后小鼠胚胎薄片的 IMPACT-Advance。我们通过研究两个相对研究较少的 PR 结构域（PRDM）蛋白 PRDM10 和 PRDM13 在不同发育阶段完整透明化小鼠胚胎中的表达，证明了这一方法的可行性。我们观察到 PRDM10 和 PRDM13 在发育中的神经系统和骨骼软骨中表达强烈，这表明这些蛋白在胚胎发生过程中在这些组织中具有功能作用。
