CELLULAR AND MOLECULAR MORPHOLOGY CORE

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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.