CELLULAR AND MOLECULAR MORPHOLOGY CORE

项目总结（见说明）： 细胞和分子形态学核心为GI研究人员提供专业知识和专业实验室设施，并为临床和基础研究开发新的测试。这个核心一直是高效和有益的TMC消化疾病中心的成员，特别是试点和可行性（P/F）获奖者，他们的经验和资源必然是有限的。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、共聚焦和去卷积显微镜、透射、扫描和免疫电子显微镜、定量形态分析、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。该中心在人类和动物组织的收集和处理方面提供咨询和培训，并向有兴趣开发复杂程序的研究人员提供技术咨询。该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力。这是通过用户团体的讲习班和会议完成的，会上介绍了新的数据、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求;以及引入光学投影断层扫描技术用于肠道发育和肠道类器官损伤的三维成像。应DDC成员要求，增加了基于成像的高通量单克隆抗体筛选。这将是一种有效的方法来产生纯化的蛋白质和抗体组的质量抗体后，“鸟枪免疫”的多种蛋白质（或细胞级分），然后在随后的反向蛋白质组学方法中使用。短地高辛标记的锁核酸（LNA）探针已通过原位核心引入。由于LNA探针比传统的核糖核酸探针（仅20-22个核苷酸长）短得多，因此它们适用于微小RNA，并且还检测RNA的特定剪接变体的细胞定位。计划包括标准化的协议，以检测多个基因在同一时间使用不同的标签标记的核糖核酸探针，并结合联合收割机RNA原位杂交和免疫组织化学。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.