CORRELATION OF EM-IMMUNOLOCALIZATION SAMPLES W/ TOMOGRAPHIC RECONSTRUCTIONS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 大分子的免疫定位是表征特定蛋白质在细胞过程中作用的任何研究的有价值的部分。然而，为获得高质量的结构保存而设计的样品制备技术往往与良好的抗体标记不兼容。此外，以高质量保存为特征的免疫标记方案(即低温Lowicryl包埋)可能不能充分揭示感兴趣的结构或对广泛的免疫学试剂有用。另一方面，通常产生精细免疫标记的技术往往会降低结构保存的质量。为了研究哺乳期大鼠乳腺上皮细胞高尔基体及其相关隔室的三维结构，我们对每个问题都采用了最佳方法，然后结合他们的结果来解决我们的科学问题。我们的结果表明，用改进的Tokuyasu方法进行免疫标记可以与高压冷冻和冷冻替代保存完好的标本的薄片和断层图像相关联。其结果是提高了蛋白质定位的准确性，并更好地了解了与乳汁分泌有关的膜结构的组织。

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.