by Ms. McHugh. Whereas most histological processing associated with Project 1 (motor neuron and neuromuscular junction counts) will also be done in this facility, additional work requiring more specialized techniques will also be performed in Dr. Mitchell's laboratory. 2. The 70% effort from Ms. McHugh may not be sufficient. We have attempted to clarify the operation of Core B. Dr. Svendsen will be coordinating overall core functions, but the individual Pi's within the program project will be responsible for interacting with Ms. McHugh. Scientists in their respective laboratories will generate the data. Thus, Ms. McHugh will not be expected to process tissue from, all three projects, but rather will provide guidance and experience necessary for the other scientists to work efficiently and accurately in meeting the goals of this program project. Thus, she should be able to manage her time efficiently and work within the 70% allowance. The remaining 30% or her time will be spent working directly with the Svendsen laboratory in Project 2. Abstract: The histology and imaging core will provide essential services to the three projects that make up this program project grant. Specifically, it will section spinal cord specimens, store sections, perform routine immunohistological stains, perform routine tracing, histological stains and establish new protocols where needed. The Core will provide for the skills and equipment necessary to perform imaging and complex stereological counts and confocal analysis of transplants in the spinal cord. This equipment is expensive and highly specialized, thus making such a shared arrangement more practical. Dr. Dan Peterson will serve as a PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page 214 Continuation Format Page Principal Investigator/Program Director (Last, First, Middle): Svendsen, Clive, N. consultant in the operation of this core, thereby adding considerable expertise pertaining to confocal and sterology applications; he will also help with the interpretation of spinal cord transplants. The experiments carried out within the program project could not be done efficiently without the services of the Histology and Immunology Core (Core B). A:
SPECIFIC AIMS The histology and imaging core will provide a common service for the three projects that comprise this proposal. The core will be closely linked with the Waisman Cellular and Molecular Neuroscience core, a facility allowing access to necessary but expensive equipment for stereology and confocal microscopy. Stereology and confocal image analysis will be vital for determining exactly how cells are surviving within the transplants, and what they have become during their in vivo maturation. Some common needs of the core have been established over the past 6 months. We have developed efficient methods for retrograde tracing, stereology, confocal microscopy and all other basic histological methods required by the projects. Having an experienced staff member to help facilitate this service will be extremely important for efficient analysis of the animals in this study, and will improve the reproducibility and comparability of data between the three projects. B: PRELIMINARY STUDIES All of the studies described in Projects 2 (Svendsen) and 3 (Zhang) were performed within the histology laboratory described here. Jackie McHugh was responsible for all of the tissue processing, immunocytochemistry and imaging analysis within the preliminary data discussed in Project 2. C: RESEARCH PLANS AND EXPERIMENTAL DESIGN The specific services to be provided by this core for the three groups are a) technical personnel proficient in tissue processing; b) provide histology and immunology reagents; c) develop new protocols; d) evaluation of transplanted cells using developed immunohistochemical protocols to visualize, GFP or BRDU and standard single, dual or triple labeling techniques for specific markers of interest including ChAT, vimentin, nestin, GFAP and human nuclei. There is generally a requirement before publication to assess exact numbers of cells that have integrated into various tissues and their state of differentiation. This can only be achieved using complex stereological and confocal methods, which are not otherwise readily available to the Pis in this program grant. While these services will be available through Core B, Ms. McHugh will be responsible in most cases for supervising individual scientists and technicians from each project-versus performing all of the sectioning/histology/analysis herself. A detailed description of the services to be provided is given below. A. Motor neuron labeling with retrograde tracer It will be important to establish that motor neurons within the spinal cord remain attached to the muscle in a number of experiments. While there are possible concerns with continued attachment, but poor transport, a Fig1. Retrograde labeling of motor neurons in the lumbar spinal cord following muscle injection of tracer. positive result with this assay will conclusively establish that the motor neurons are still projecting to the muscle. Core B will provide the investigator with the tracer and coordinates for transplantation. Proof of PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page 215 Continuation Format Page
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