Department of Medical Oncology and Hematology, University of Zurich and University Hospital Zurich, Zurich, Switzerland.
Methods Mol Biol. 2021;2308:127-138. doi: 10.1007/978-1-0716-1425-9_11.
The soft marrow tissues, which are found disseminated throughout bone cavities, are prime sites for hematopoietic cell production, development, and control of immune responses, and regulation of skeletal metabolism. These essential functions are executed through the concerted and finely tuned interaction of a large variety of cell types of hematopoietic and nonhematopoietic origin, through yet largely unknown sophisticated molecular mechanisms. A fundamental insight of the biological underpinnings of organ function can be gained from the microscopic study of the bone marrow (BM), its complex structural organization and the existence of cell-specific spatial associations. Albeit the application of advanced imaging techniques to the analysis of BM has historically proved challenging, recent technological developments now enable the interrogation of organ-wide regions of marrow tissues in three dimensions at high resolution. Here, we provide a detailed experimental protocol for the generation of thick slices of BM from murine femoral cavities, the immunostaining of cellular and structural components within these samples, and their optical clearing, which enhances the depth at which optical sectioning can be performed with standard confocal microscopes. Collectively, the experimental pipeline here described allows for the rendering of single-cell resolution, multidimensional reconstructions of vast volumes of the complex BM microenvironment.
骨髓软组织广泛分布于骨腔中，是造血细胞生成、发育以及控制免疫反应和调节骨骼代谢的主要部位。这些基本功能是通过大量不同来源的造血细胞和非造血细胞类型的协同和精细调节的相互作用来执行的，其涉及的分子机制目前尚不清楚。从骨髓（BM）的微观研究中可以深入了解器官功能的生物学基础，包括其复杂的结构组织和细胞特异性空间关联的存在。尽管将先进的成像技术应用于 BM 的分析在历史上一直具有挑战性，但最近的技术发展现在能够以高分辨率在三维空间中对骨髓组织的全器官区域进行询问。在这里，我们提供了一个从鼠股骨腔中生成 BM 厚片的详细实验方案，包括对这些样本中细胞和结构成分的免疫染色，以及对其进行光学透明化处理，这可以增强使用标准共聚焦显微镜进行光学切片的深度。总的来说，这里描述的实验方案可以实现单细胞分辨率，对复杂的 BM 微环境的大量体积进行多维重建。
