Morphology Support

形态学支持

基本信息

批准号：
7753054
负责人：
David S Milstone
金额：
$ 30.15万
依托单位：
BRIGHAM AND WOMEN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-07-01 至 2014-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7753054
关键词：
Acute Adhesions American Anatomy Animals Biological Models Cells Cellular biology Chronic Collaborations Communication Complex Mixtures Consultations Cultured Cells Detection Educational Status Electron Microscopy Electrons Endothelium Epitopes Event Functional disorder Gene Expression Goals Histology Hospitals Human Resources Image Analysis In Situ Hybridization Individual Inflammation Injury Leukocytes Light Messenger RNA Methods Microscopic Modeling Molecular Monoclonal Antibodies Morphology Nature Normal tissue morphology Nucleic Acid Probes Organ Culture Techniques Pathologist Pathology Physiological Preparation Procedures Process Proteins Protocols documentation Publications RNA Reagent Regulation Research Personnel Scientific Advances and Accomplishments Sensitivity and Specificity Specimen Staining method Stains System Technical Expertise Techniques Testing Tissues Titrations Training Woman cytokine experience human tissue in vitro Model in vivo medical schools molecular imaging prevent programs research study success

项目摘要

The primary goal of Morphology Support is to make available to the investigators of individual projects the expertise, facilities, techniques and technical support required for morphologic, immunolocalization and gene expression studies of cultured cells and animal and human tissues at both the light and electron microscopic level. The Core supervisor. Dr. Milstone, is an American Board of Pathology certified anatomic pathologist and molecular experimental pathologist with a high level of training and expertise. He will provide consultation to investigators within the Program on interpreting histopathologic changes and identifying optimal procedures for selecting and preparing cells and tissues of experimental animals for morphologic studies. During the renewal period the Program will continue to expand utilization of in vivo and in vitro model systems and reagents, many developed previously within this Program or to be developed in the renewal period, to investigate molecular regulation and consequences of endothelial-dependent physiologic and pathophysiologic processes. Many of these experimental systems test the effects of under- or over-expressing critical adhesion, activation and/or interaction molecules or cytokines in othenwise "normal" tissues and in models of acute and chronic inflammation and tissue injury. Interpreting these experimental results requires describing reliably the morphologic changes, including assessing the degree and nature of leukocyte influx and resultant tissue injury, and immunocytochemical localization of the molecules in question. An important function of Morphology Support will therefore be providing consistent, high quality histology and sensitive methods for immunocytochemical localization. Program studies of intracellular communication events and cell biology will require expanded ultrastructural analysis by electron microscopy and perhaps immunoelectronmicroscopy. Over several Project periods Morphology Support has developed considerable experience in these techniques. The Core benefits from dedicated and well-trained technical personnel who have performed such analyses for up to twenty years in support ofthe Projects of this Program. They understand the various techniques required, the needs of individual investigators and many of the scientific issues particular to individual projects. Morphology Support has developed protocols that optimize sensitivity and specificity of immunocytochemical localization, as evidenced by numerous publications emanating from this Program during several previous Program periods. Examples are titration of monoclonal antibodies to allow detecting increased or decreased abundance of a molecule which is present in the basal state (Coxon, A et al., 2001, Delfs, M W et al., 2001, Milstone, D S et al., 2000, Milstone, D S et al., 2000), adaptation of monoclonal antibodies generated against molecules in one experimental species to use in a different species (Milstone, D S et al., 2001), simultaneous double staining with two monoclonal antibodies to distinguish two different protein epitopes in a single tissue section, and immunolocalization in organ culture. Morphology Support has also successfully developed RNA in situ hybridization that requires additional expertise in handling target specimens and molecularly cloned nucleic acid probes to prevent degradation by RNAses present in target cells and tissues, and by exogenous contaminant RNAses. Extensive expertise by Dr. Milstone in preparing specimens and probes for specific detection of small quantities of mRNA in complex mixtures has supported success of this approach. Colleagues with experience in this technique are also available within the Departments of Pathology of Brigham and Women's Hospital and Harvard Medical School, if needed. These techniques carefully developed, validated and performed by Morphology Support personnel will provide to Program Investigators morphologic and immunohistochemical preparations appropriate for image analysis. Dr. Lichtman in Project 2 has successfully accomplished this in the past using preparations from Morphology Support. Personnel in Core C: Physiological and Molecular Imaging Support of this Program, supervised by Dr. Tanya Mayadas will perform these analyses in the renewal period. The technical expertise of both Cores will thus synergize to advance the scientific goals ofthe Program and its individual components

形态支持的主要目标是向单个项目的调查人员提供形态学，免疫定位和基因所需的专业知识，设施，技术和技术支持在光和电子显微镜下培养的细胞以及动物和人体组织的表达研究等级。核心主管。 Milstone博士是美国病理学委员会认证的解剖病理学家，具有高水平的培训和专业知识的分子实验病理学家。他将向该计划中的调查人员解释组织病理学变化并确定最佳程序选择和制备实验动物的细胞和组织进行形态学研究。在续签期间，该计划将继续扩大体内和体外模型系统的利用率和试剂，许多试剂以前在此程序中开发或在续签期间开发，研究内皮依赖性生理和病理生理的分子调节和后果过程。这些实验系统中的许多都测试了不足或过度表达的临界粘附的影响，在Othenwise的“正常”组织中的激活和/或相互作用分子或细胞因子以及急性和急性模型中慢性炎症和组织损伤。解释这些实验结果需要可靠地描述形态学的变化，包括评估白细胞流入的程度和性质和由此导致的组织损伤，以及所讨论的分子的免疫细胞化学定位。形态学的重要功能因此，支持将为一致，高质量的组织学和敏感方法提供免疫细胞化学定位。细胞内通信事件和细胞生物学的计划研究将需要通过电子显微镜和可能的免疫电子显微镜进行扩展的超微结构分析。在几个项目时期，形态支持在这些技术方面已经发展了丰富的经验。敬业和训练有素的技术人员的核心好处，他们已经进行了此类分析长达20年来支持该计划的项目。他们了解所需的各种技术，个别调查人员的需求以及各个项目的许多科学问题。形态支持开发了优化免疫细胞化学敏感性和特异性的方案本地化，这是在以前的几个计划中发出的许多出版物所证明的计划期限。例如，单克隆抗体的滴定允许检测到增加或减少存在于基础状态的分子的丰度（Coxon，A等，2001，Delfs，M W等，2001， Milstone，D S等人，2000年，Milstone，D S等，2000），针对针对的单克隆抗体的适应一个实验物种中用于不同物种的分子（Milstone，D S等，2001），同时用两种单克隆抗体进行双重染色，以区分单个组织中的两个不同的蛋白质表位部分和人体培养中的免疫定位。形态支持还成功地开发了RNA原位杂交，需要额外处理靶标本和分子克隆的核酸探针方面的专业知识，以防止通过靶细胞和组织以及外源污染物RNass中存在的RNass。广泛的专业知识 Milstone博士在准备标本和探针中，以特异性检测复合物中的少量mRNA 混合物支持了这种方法的成功。具有此技术经验的同事也是在杨百翰和妇女医院和哈佛医学院病理学系中可用如果需要。这些技术由形态支持人员精心开发，验证和执行，将提供为研究人员编程适合图像分析的形态和免疫组织化学制剂。 Lichtman博士在项目2中已成功地使用了形态的准备工作来实现这一目标支持。 C核C中的人员：该程序的生理和分子成像支持，由博士指导 Tanya Mayadas将在续签期间进行这些分析。两个核心的技术专长将因此协同促进了该计划的科学目标及其各个组成部分