The primary goal of Morphology Support is to make available to the investigators of individual projects the expertise, facilities, techniques and technical support required for morphologic, immunolocalization and gene expression studies of cultured cells and animal and human tissues at both the light and electron microscopic level. The Core supervisor. Dr. Milstone, is an American Board of Pathology certified anatomic pathologist and molecular experimental pathologist with a high level of training and expertise. He will provide consultation to investigators within the Program on interpreting histopathologic changes and identifying optimal procedures for selecting and preparing cells and tissues of experimental animals for morphologic studies. During the renewal period the Program will continue to expand utilization of in vivo and in vitro model systems and reagents, many developed previously within this Program or to be developed in the renewal period, to investigate molecular regulation and consequences of endothelial-dependent physiologic and pathophysiologic processes. Many of these experimental systems test the effects of under- or over-expressing critical adhesion, activation and/or interaction molecules or cytokines in othenwise """"""""normal"""""""" tissues and in models of acute and chronic inflammation and tissue injury. Interpreting these experimental results requires describing reliably the morphologic changes, including assessing the degree and nature of leukocyte influx and resultant tissue injury, and immunocytochemical localization of the molecules in question. An important function of Morphology Support will therefore be providing consistent, high quality histology and sensitive methods for immunocytochemical localization. Program studies of intracellular communication events and cell biology will require expanded ultrastructural analysis by electron microscopy and perhaps immunoelectronmicroscopy. Over several Project periods Morphology Support has developed considerable experience in these techniques. The Core benefits from dedicated and well-trained technical personnel who have performed such analyses for up to twenty years in support ofthe Projects of this Program. They understand the various techniques required, the needs of individual investigators and many of the scientific issues particular to individual projects. Morphology Support has developed protocols that optimize sensitivity and specificity of immunocytochemical localization, as evidenced by numerous publications emanating from this Program during several previous Program periods. Examples are titration of monoclonal antibodies to allow detecting increased or decreased abundance of a molecule which is present in the basal state (Coxon, A et al., 2001, Delfs, M W et al., 2001, Milstone, D S et al., 2000, Milstone, D S et al., 2000), adaptation of monoclonal antibodies generated against molecules in one experimental species to use in a different species (Milstone, D S et al., 2001), simultaneous double staining with two monoclonal antibodies to distinguish two different protein epitopes in a single tissue section, and immunolocalization in organ culture. Morphology Support has also successfully developed RNA in situ hybridization that requires additional expertise in handling target specimens and molecularly cloned nucleic acid probes to prevent degradation by RNAses present in target cells and tissues, and by exogenous contaminant RNAses. Extensive expertise by Dr. Milstone in preparing specimens and probes for specific detection of small quantities of mRNA in complex mixtures has supported success of this approach. Colleagues with experience in this technique are also available within the Departments of Pathology of Brigham and Women's Hospital and Harvard Medical School, if needed. These techniques carefully developed, validated and performed by Morphology Support personnel will provide to Program Investigators morphologic and immunohistochemical preparations appropriate for image analysis. Dr. Lichtman in Project 2 has successfully accomplished this in the past using preparations from Morphology Support. Personnel in Core C: Physiological and Molecular Imaging Support of this Program, supervised by Dr. Tanya Mayadas will perform these analyses in the renewal period. The technical expertise of both Cores will thus synergize to advance the scientific goals ofthe Program and its individual components
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