CELLULAR AND MOLECULAR MORPHOLOGY CORE

项目总结(见说明)： 细胞和分子形态核心为GL研究人员提供专业知识和专门的实验室设施，并为临床和基础研究开发新的测试。这一核心对TMC消化疾病中心的成员，特别是试点和可行性(P/F)奖获得者来说，具有很高的生产力和益处，他们的经验和资源必然有限。在过去的4年里，50名正式成员中有22名使用了这一核心，79名准成员和P/F获奖者中有16名使用了这一核心。主要服务包括组织学、免疫组织化学、原位杂交、酶组织化学和免疫荧光抗体研究、共聚焦显微镜和去卷积显微镜、透射、扫描和免疫电子显微镜、定量形态计量分析、用于分子遗传分析的激光捕获解剖，以及用于互联网通信和出版的数字图像。该中心提供人类和动物组织收集和加工方面的咨询和培训，并为有兴趣开发复杂程序的研究人员提供技术建议。这一核心的一个主要好处是共享从各个实验室的不同项目中获得的信息，以提高所有实验室的生产率。这是通过举办讲习班和用户小组会议来实现的，在这些会议上介绍了新的数据、方法和程序。过去4年的进展包括：升级激光捕获显微镜系统，用于相位和荧光显微镜；增加40种新抗体，其中一些具有双重染色，以满足研究人员的需要；以及引入光学投影断层扫描技术，用于肠道器官发育和损伤的三维成像。应DDC成员的要求，增加了基于高通量成像的单抗筛选。这将是一种有效的方法，在对多个蛋白质(或细胞部分)进行‘鸟枪式免疫’后，产生针对纯化蛋白质和抗体面板的高质量抗体，然后将其用于后续的反向蛋白质组学方法。原位核已经引入了短的地高辛标记的锁定核酸(LNA)探针。由于它们比传统的核探针(只有20-22个核苷酸长)短得多，所以LNA探针适用于微小RNA，还可以检测RNA的特定剪接变体的细胞定位。计划包括标准化一项协议，使用标记了不同标签的核探针一次检测多个基因，并将RNA原位杂交和免疫组织化学结合起来。这将用荧光探针进行测试，以允许共聚焦显微镜进行精确的细胞定位。

PROJECT SUMMARY (See instructions): The Cellular and Molecular Morphology Core provides expertise and specialized laboratory facilities to Gl researchers and develops new tests for clinical and basic research. This Core has been highly productive and beneficial for members of the TMC Digestive Diseases Center, particularly to Pilot and Feasibility (P/F) awardees, whose experience and resources are necessarily limited. In the last 4 years, 22 of the 50 Full Members used this Core as did 16 of 79 Associate members and P/F Awardees. Major services include histology, immunohistochemistry, in-situ hybridization, enzyme histochemistry and immunofluorescent antibody studies, confocal and deconvolution microscopy, transmission, scanning, and immunoelectron microscopy, quantitative morphometric analysis, laser capture dissection for molecular genetic analyses, and digital images for internet communication and publication. The Core provides consultation and training in collection and processing of human and animal tissues, as well as technical advice to researchers interested in developing sophisticated procedures. A major benefit of this Core is the sharing of information gained from diverse projects in individual laboratories to enhance the productivity of all. This is accomplished by workshops and meetings of the user groups at which new data, methods and procedures are presented. Advances during the past 4 years include: upgrading of the Laser Capture Microscopy system for phase and fluorescence microscopy; adding 40 new antibodies, some with double staining to meet investigators' needs; and the introduction of Optical Projection Tomography for 3-dimensional imaging of intestinal development and injury of intestinal organoids. High throughput imaging-based Monoclonal Antibody Screening was added in response to DDC members requests. This will be an efficient way to generate quality antibodies to purified proteins and antibody panels following 'shotgun immunizations' of multiple proteins (or cell fractions) which are then used in subsequent reverse proteomics approaches. Short DIG-labeled locked nucleic acid (LNA) probes have been introduced by the In-Situ Core. Since they are much shorter than conventional riboprobes (only 20-22 nucleotides long), LNA probes are applicable to micro RNAs and also detect the cellular localization of specific splice variants of RNA. Plans include standardization of a protocol to detect multiple genes at a time using riboprobes labeled with different tags and to combine RNA in situ hybridization and immunohistochemistry. This will be tested With fluorescent probes to permit confocal microscopy for exquisite cellular localization.