Ni Yuehan, Wu Jiamiao, Liu Fengqi, Yi Yating, Meng Xiangjiao, Gao Xiang, Xiao Luyi, Zhou Weiwei, Chen Zexi, Chu Peng, Xing Dan, Yuan Ye, Ding Donghui, Shen Ge, Yang Min, Wu Ronjie, Wang Ling, Melo Luiza Martins Nascentes, Lin Sien, Cheng Xiaoguang, Li Gang, Tasdogan Alpaslan, Ubellacker Jessalyn M, Zhao Hu, Fang Shentong, Shen Bo
College of Life Sciences, Beijing Normal University, 100875, Beijing, China.
National Institute of Biological Sciences, Beijing (NIBS), 102206, Beijing, China.
Bone Res. 2025 Jan 13;13(1):6. doi: 10.1038/s41413-024-00387-9.
Tissue clearing combined with high-resolution confocal imaging is a cutting-edge approach for dissecting the three-dimensional (3D) architecture of tissues and deciphering cellular spatial interactions under physiological and pathological conditions. Deciphering the spatial interaction of leptin receptor-expressing (LepR) stromal cells with other compartments in the bone marrow is crucial for a deeper understanding of the stem cell niche and the skeletal tissue. In this study, we introduce an optimized protocol for the 3D analysis of skeletal tissues, enabling the visualization of hematopoietic and stromal cells, especially LepR stromal cells, within optically cleared bone hemisections. Our method preserves the 3D tissue architecture and is extendable to other hematopoietic sites such as calvaria and vertebrae. The protocol entails tissue fixation, decalcification, and cryosectioning to reveal the marrow cavity. Completed within approximately 12 days, this process yields highly transparent tissues that maintain genetically encoded or antibody-stained fluorescent signals. The bone hemisections are compatible with diverse antibody labeling strategies. Confocal microscopy of these transparent samples allows for qualitative and quantitative image analysis using Aivia or Bitplane Imaris software, assessing a spectrum of parameters. With proper storage, the fluorescent signal in the stained and cleared bone hemisections remains intact for at least 2-3 months. This protocol is robust, straightforward to implement, and highly reproducible, offering a valuable tool for tissue architecture and cellular interaction studies.
组织透明化结合高分辨率共聚焦成像技术是一种前沿方法，用于剖析组织的三维（3D）结构，并解读生理和病理条件下的细胞空间相互作用。解读表达瘦素受体（LepR）的基质细胞与骨髓中其他区室的空间相互作用，对于深入理解干细胞生态位和骨骼组织至关重要。在本研究中，我们介绍了一种用于骨骼组织三维分析的优化方案，能够在光学透明的骨半切片中可视化造血细胞和基质细胞，特别是LepR基质细胞。我们的方法保留了三维组织结构，并且可扩展到其他造血部位，如颅骨和椎骨。该方案包括组织固定、脱钙和冷冻切片以显示骨髓腔。这个过程大约在12天内完成，产生高度透明的组织，这些组织能保持基因编码或抗体染色的荧光信号。骨半切片与多种抗体标记策略兼容。对这些透明样本进行共聚焦显微镜检查，可以使用Aivia或Bitplane Imaris软件进行定性和定量图像分析，评估一系列参数。经过适当储存，染色并透明化的骨半切片中的荧光信号至少可完整保留2至3个月。该方案稳健、易于实施且具有高度可重复性，为组织结构和细胞相互作用研究提供了一个有价值的工具。
