Multicolor Cell Membrane Profiling For Development And Disease

描述：用亲脂性碳菁染料追踪神经元连接已经彻底改变了神经解剖束追踪，是了解对照和突变小鼠大脑连接发育的基本特征，特别是因为多种颜色的染料可以使用。最近，亲脂性染料在血管标记方面也取得了进展，通过直接染色接触内皮细胞膜。阻碍这些非常成功的染料更广泛使用的一个长期存在的问题是，它们与其他程序（如免疫染色或详细的组织学）的结合有限，这些程序需要脱水才能嵌入，因为染料分子不能永久地与膜结合，可能会泄漏或容易被亲脂溶剂或洗涤剂冲走。迄今为止，克服这些问题的努力最多只取得了部分成功。因此，为了进一步扩大碳菁亲脂染料的使用，我们建议开发可固定的碳菁染料，它可以与膜结合蛋白中的赖氨酸基团结合，从而即使在脂质双层被洗涤剂或有机溶剂去除后也能保留在膜上。我们计划通过开发一种方案来优化这些染料的使用，该方案允许组合多个不同的荧光团，以最大限度地从给定的模式生物中获得信息。具体来说，我们的具体目标是：(1)合成四种光谱上不同的可固定的亲脂性染料，三种用于神经束追踪，一种用于血管标记，与组织处理和免疫细胞化学方案中使用的标准固定技术兼容。合成的染料将具有芳香的n -羟基琥珀酰亚胺酯基团，为膜蛋白提供共价锚定。(2)使用光学显微镜水平上高分辨率组织学所需的处理技术，在固定组织的标准化测试系统中评估可固定染料。在这个目标中，我们将描述这些染料在用有机溶剂和洗涤剂处理后在组织中保留的时间，以及随着时间的推移洗涤的过程。我们还将描述这些染料相互结合的最佳条件，以及与免疫化学和/或垂死细胞染色的最佳条件。(3)创建测试产品，将每种染料单独或组合销售给研究界。我们将开发测试产品，并将其发送给神经科学界的5-10名合作者，以及在SA2中开发的已发布协议。结合起来，这三个目标将为与血脑屏障破坏相关的研究提供有用的新试剂，如肿瘤、创伤和神经退行性疾病以及正常发育。

DESCRIPTION: Tracing neuronal connections with lipophilic carbocyanine dyes has revolutionized neuroanatomical tract tracing and is an essential feature to understand development of brain connections in both control and mutant mice in particular since multiple colors of dyes can be used. More recently, lipophilic dyes have also gained ground in labeling blood vessels by directly staining the endothelial cell membranes upon contact. A persistent problem that blocks even wider use of these extremely successful dyes is their limited combination with other procedures such as immunostaining or detailed histology that requires dehydration for embedding, since the dye molecules are not permanently bound to the membranes and can either leak out or be easily washed away by lipophilic solvents or detergents. Attempts to overcome these problems have thus far been at best partially successful. Therefore, to broaden even further the use of carbocyanine lipophilic dyes we propose to develop fixable carbocyanine dyes that can be bonded to lysine groups in membrane bound proteins thereby retaining the dyes in the membranes even after the lipid bilayers has been removed with detergents or organic solvents. We plan to optimize the use of these dyes by developing a protocol that allows combination of multiple distinct fluorophores to maximize the information gained from a given model organism. Specifically, our Specific Aims are: (1) Synthesize four spectrally distinct fixable lipophilic dyes, three for nerve tract tracing and one or blood vessel labeling, that are compatible with standard fixation techniques used in tissue processing and immunocytochemistry protocols. Dyes synthesized will feature an aromatic N-hydroxysuccinimide ester group to provide covalent anchoring to membrane proteins. (2) Evaluate fixable dyes in standardized test systems in fixed tissue using processing techniques needed for high resolution histology at the light microscope level. In this aim we will characteriz how long these dyes are retained in tissue after treatment with organic solvents and detergents and how washing out progresses over time. We will also characterize the conditions under which these dyes can be best combined with each other and with immunochemistry and/or staining for dying cells. (3) Create test products to market each dye alone or in combination to the research community. We will develop test products to be sent to 5-10 collaborators in the neuroscience community along with the published protocol developed in SA2. Combined, these three aims will provide novel reagents useful for studies related to breaches in the blood-brain barrier such as tumors, trauma and neurodegenerative diseases as well as normal development.