Bryson Jerrod L, Coles Mark C, Manley Nancy R
Department of Cellular Biology, University of Georgia, USA.
J Vis Exp. 2011 Oct 7(56):3267. doi: 10.3791/3267.
The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in the thymus proper vasculature formation and patterning is essential for thymocyte entry into the organ and mature T-cell exit to the periphery. The spatial arrangement of blood vessels in the thymus is dependent upon signals from the local microenvironment, namely thymic epithelial cells (TEC). Several recent reports suggest that disruption of these signals results in thymus blood vessel defects. Previous studies have described techniques used to label the neonatal and adult thymus vasculature. We demonstrate here a technique for labeling blood vessels in the embryonic thymus. This method combines the use of FITC-dextran or Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL 1-isolectin B₄) facial vein injections and CD31 antibody staining to identify thymus vascular structures and PDGFR-β to label thymic perivascular mesenchyme. The option of using cryosections or vibratome sections is also provided. This protocol can be used to identify thymus vascular defects, which is critical for defining the roles of TEC-derived molecules in thymus blood vessel formation. As the method labels the entire vasculature, it can also be used to analyze the vascular networks in multiple organs and tissues throughout the embryo including skin and heart.
功能性血管网络的建立是器官发生的重要组成部分，也是器官最佳功能所必需的。例如，在胸腺中，正常的脉管系统形成和模式对于胸腺细胞进入器官以及成熟T细胞向外周输出至关重要。胸腺中血管的空间排列取决于局部微环境的信号，即胸腺上皮细胞（TEC）。最近的几份报告表明，这些信号的破坏会导致胸腺血管缺陷。先前的研究描述了用于标记新生和成年胸腺脉管系统的技术。我们在此展示一种用于标记胚胎胸腺血管的技术。该方法结合使用异硫氰酸荧光素 - 葡聚糖或 Griffonia（Bandeiraea）Simplicifolia凝集素I（GSL 1 - 异凝集素B₄）经面静脉注射以及CD31抗体染色来识别胸腺血管结构，并使用血小板衍生生长因子受体β（PDGFR - β）来标记胸腺血管周围间充质。还提供了使用冰冻切片或振动切片的选择。该方案可用于识别胸腺血管缺陷，这对于确定TEC衍生分子在胸腺血管形成中的作用至关重要。由于该方法标记了整个脉管系统，它还可用于分析整个胚胎中包括皮肤和心脏在内的多个器官和组织中的血管网络。
