Multicolor Cell Membrane Profiling For Development And Disease

产品说明：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学束追踪，并且特别是因为可以使用多种颜色的染料，所以是理解对照和突变小鼠中脑连接发育的基本特征。最近，亲脂性染料也通过在接触时直接染色内皮细胞膜而在标记血管中获得了基础。阻碍这些非常成功的染料的更广泛使用的一个持续存在的问题是它们与其他程序的有限组合，例如免疫染色或需要脱水以进行包埋的详细组织学，因为染料分子不能永久地结合到膜上，并且可以泄漏或容易地被亲脂性溶剂或洗涤剂洗掉。迄今为止，克服这些问题的努力充其量只是部分成功。因此，为了进一步拓宽碳菁亲脂性染料的用途，我们提出开发可固定的碳菁染料，其可以与膜结合蛋白质中的赖氨酸基团键合，从而即使在用洗涤剂或有机溶剂除去脂质双层之后也将染料保留在膜中。我们计划通过开发一种协议来优化这些染料的使用，该协议允许多个不同的荧光团的组合，以最大限度地提高从给定的模式生物中获得的信息。 具体而言，我们的具体目标是：（1）合成四种光谱不同的可固定亲脂性染料，三种用于神经束示踪，一种用于血管标记，其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族N-羟基琥珀酰亚胺酯基团，以提供与膜蛋白的共价锚定。(2)使用光学显微镜水平下高分辨率组织学所需的处理技术，在标准化测试系统中对固定组织中的可固定染料进行评价。在这个目标中，我们将表征这些染料在用有机溶剂和洗涤剂处理后在组织中保留多长时间，以及随着时间的推移如何洗掉。我们还将表征这些染料可以最好地相互结合以及与免疫化学和/或染色垂死细胞的条件。(3)创建测试产品，将每种染料单独或组合销售给研究社区。我们将开发测试产品，并将其发送给神经科学界的5-10名合作者沿着在SA 2中开发的已发布方案。 这三个目标相结合，将提供新的试剂，可用于与血脑屏障破坏相关的研究，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.