CORRELATION OF EM-IMMUNOLOCALIZATION SAMPLES W/ TOMOGRAPHIC RECONSTRUCTIONS

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 大分子的免疫定位是任何表征特定蛋白质在细胞过程中的作用的研究的有价值的部分。 然而，设计用于产生高质量结构保留的样品制备技术通常与良好的抗体标记不相容。 此外，以高质量保存为特征的免疫标记方案（即低温Lowicryl包埋）可能无法充分揭示感兴趣的结构或适用于广泛的免疫试剂。 另一方面，通常产生精细免疫标记的技术通常导致结构保存质量降低。为了研究哺乳期大鼠乳腺上皮细胞中高尔基体和相关隔室的3D结构，我们采用了针对每个问题的最佳方法，然后将其结果联合收割机用于解决我们的科学问题。 我们的研究结果表明，免疫标记与修改的Tokuyasu方法可以与保存完好的标本制备的高压冷冻和冷冻置换的薄切片和断层图像。 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.