CORRELATION OF EM-IMMUNOLOCALIZATION SAMPLES W/ TOMOGRAPHIC RECONSTRUCTIONS

电磁免疫定位样本与断层扫描重建的相关性

• 批准号: 7354974
• 负责人: MARK S LADINSKY
• 金额: $ 1.41万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-26 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7354974
• 关键词: antibody; cold temperature; freeze substitution; hand; lactates; macromolecule; membrane structure; milk; motivation; protein localization; proteins; role; secretion; sectioning; antibody; cold temperature; freeze substitution; hand; lactates; macromolecule; membrane structure; milk; motivation; protein localization; proteins; role; secretion; sectioning

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Immunolocalization of macromolecules is a valuable part of any study that characterizes the roles of specific proteins in cellular processes. However, sample preparation techniques that are designed to yield high quality structural preservation are often not compatible with good antibody labeling. Moreover, immunolabeling protocols that do feature high quality preservation (i.e. low-temperature Lowicryl embedding) may not adequately reveal structures of interest or be useful for a broad range of immunological reagents. On the other hand, techniques that usually yield fine immunolabeling often result in a reduced quality of structural preservation. To study the 3D structure of the Golgi and associated compartments in lactating rat mammary epithelial cells, we have employed the best approaches for each of these issues and then combine their results to address our scientific questions. Our results demonstrate that immunolabeling with a modification of the Tokuyasu method can be correlated with thin-section and tomographic images from well-preserved specimens prepared by high-pressure freezing and freeze-substitution. The result is an improvement in the accuracy of the localization of proteins and greater understanding of the organization of membrane structures involved in milk secretion.

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子弹和调查员（PI）可能已经从其他NIH来源获得了主要资金，因此可以在其他清晰的条目中代表。列出的机构适用于该中心，这不一定是调查员的机构。大分子的免疫定位是表征特定蛋白在细胞过程中作用的任何研究的重要组成部分。但是，旨在产生高质量结构保存的样品制备技术通常与良好的抗体标记不兼容。此外，确实具有高质量保存的免疫标记方案（即低温Lowicryl嵌入）可能无法充分揭示感兴趣的结构，也可能对广泛的免疫试剂有用。另一方面，通常会产生精细免疫标记的技术通常会导致结构保存的质量降低。为了研究高尔基体的3D结构和在泌乳大鼠乳腺上皮细胞中的隔室，我们采用了这些问题的最佳方法，然后结合其结果来解决我们的科学问题。我们的结果表明，通过对Tokuyasu方法进行修饰的免疫标记可以与通过高压冻结和冻结替代制备的薄片和层析成像图像相关联。结果是提高了蛋白质定位的准确性以及对参与牛奶分泌的膜结构的组织的更多了解。