Multicolor Cell Membrane Profiling For Development And Disease

发育和疾病的多色细胞膜分析

基本信息

批准号：
8589954
负责人：
Brian David Gray
金额：
$ 21.99万
依托单位：
MOLECULAR TARGETING TECHNOLOGIES, INC.
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-01 至 2015-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8589954
关键词：
Aftercare Aldehydes Amides Animal Model Apoptotic Axon Binding Binding Proteins Blood - brain barrier anatomy Blood Vessels Blood capillaries Brain Brain Diseases Brain Neoplasms Carbocyanines Cell Death Cell Membrane Proteins Cell membrane Cells Collaborations Color Communities Dehydration Detergents Development Diffusion Disease Disease model Dyes Embryo Endothelial Cells Esters Ethanol Feedback Fetal Alcohol Syndrome Fiber Fluorescent Dyes Generations Gray unit of radiation dose High Pressure Liquid Chromatography Histology Image Imaging Techniques Immune Immunochemistry Individual Iowa Knowledge Label Labyrinth Lateral Life Light Microscope Lipid Bilayers Lysine Marketing Membrane Membrane Proteins Molecular Target Motor Neurons Mutant Strains Mice Nerve Neuroanatomy Neurodegenerative Disorders Neurons Neurosciences Oranges Organic solvent product Performance Phase Population Preservation Technique Procedures Process Property Protocols documentation Publishing Reagent Research Research Personnel Resolution Sensory Small Business Innovation Research Grant Solid Solutions Solvents Specimen Staining method Stains System Techniques Technology Temperature Testing Time Tissue Preservation Tissues Tracer Trauma Universities Variant Vascular blood supply Work amino group analog capillary fluorescence imaging fluorophore immunocytochemistry improved meetings mutant novel public health relevance sample fixation tissue fixing tissue processing tumor

项目摘要

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.

描述：通过亲脂性碳酸染料来追踪神经元连接已革新神经解剖学跟踪，并且是了解控制和突变小鼠脑连接发展的重要特征，因为可以使用多种染料的染料。最近，亲脂性染料在接触时通过直接染色的内皮细胞膜染色，在标记血管方面也获得了基础。一个持久的问题，即将阻止这些极为成功的染料更广泛地使用这些染料，它们与其他程序（例如免疫染色或详细的组织学）的组合有限，因为染料分子没有永久绑定到膜上，并且可以泄漏或被亲脂性溶剂渗出，因为染料分子没有永久绑定到膜上。迄今为止，试图克服这些问题的尝试最多部分成功。因此，为了进一步扩大使用碳酸亲脂性染料的使用，我们建议开发可固定的碳酸染料，这些染料可以与膜结合蛋白中的赖氨酸基团粘合到膜结合的蛋白中，从而将膜中的染料保留在脂质双层后，与洗涤剂或有机溶剂一起去除脂质双层。我们计划通过制定允许多个不同不同荧光团结合的协议来优化这些染料的使用，以最大程度地利用从给定模型生物那里获得的信息。具体而言，我们的具体目的是：（1）合成四种频谱固定的亲脂性染料，三个用于神经道跟踪的染料和一个或血管标记，它们与在组织加工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族N-羟基糖二酰亚胺酯基，以提供共价锚固到膜蛋白。（2）使用在光学显微镜水平上高分辨率组织学所需的处理技术评估固定组织中标准化测试系统中的可固定染料。在此目标中，我们将表征这些染料在用有机溶剂和洗涤剂处理后将这些染料保留在组织中的时间，以及随着时间的推移如何洗涤。我们还将表征这些染料可以最好地相互结合的条件，并通过免疫化学和/或染色用于垂死的细胞。（3）创建测试产品以单独或将每种染料销售给研究社区。我们将开发测试产品，将与SA2中开发的已发表协议一起发送给神经科学社区的5-10个合作者。这三个目标结合在一起，将为与血脑屏障，例如肿瘤，创伤和神经退行性疾病以及正常发育相关的研究提供新的试剂。