Provides equipment, methodology consultation, and reagents that assist in the introduction of transgenes for transient transfection, and in the localization of transgene and endogenous gene products. The most commonly used services are listed below, but, depending on investigator interest, other methods also can be offered as collaborative services.
Basic molecular biology and histology techniques
Techniques include, but are not limited to, animal perfusions, long-term preservation of tissue specimens, brain dissections, nucleic acids isolation and quantification, gel electrophoresis, Northern and Southern Blots, cellular fractionation, PCR and Western Blots. While these are routine methods in most laboratories, core equipment and experience help investigators devise, perform, and validate assays.
Expertise is provided for thionin/Nissl staining, widely used for revealing cell bodies and cytoarchitecture of brain areas, and for acetylcholinesterase staining, used to identify cholinergic neurons and fibers. Assistance with Hematoxylin and Eosin (cells, cytoarchiatecture), FluoroJade (degenerating cells), and Luxol Fast blue (myelin) staining also are available.
Antibodies are used to detect the presence of specific molecules (antigens) in tissue sections and provide the unique distribution pattern for a given antigen. IHC is performed both on slide-mounted sections or/and on fixed, free-floating sections, which also can be generated by the Core’s equipment. Core personnel have expertise with staining for >50 proteins, including, but not limited to transcription factors, neurotransmitter synthesis pathways, receptors, glial markers, cell adhesion proteins, inflammatory markers, cell signaling components, and synaptic proteins.
qPCR primer design and validation
Thoughtful primer design and validation are essential to ensure a sensitive and specific assay. The Core assists investigators in developing optimal qPCR primer sets for their needs. Once the assay is optimized, the investigator will perform the qPCR experiments in the Vanderbilt Technologies for Advanced Genomics Core (VANTAGE). http://vantage.vanderbilt.edu
Cell labeling with lipophillic dyes using Gene Gun
The BioRad Gene Gun is used to label cells with lipophilic dyes for analysis of dendritic and axonal arbors. Particles are coated with fluorescent dyes, and “shot in” into the cells of interest. Inside the cell, the dyes are dissolved from the surface of the particles and visualize the cellular arborization.
Intrauterine electroporation of transgenes into mouse embryos
Electroporation of selected genes into the brain of developing embryos is a powerful tool, but in addition to special instrumentation, requires hands-on training, which the Core provides.
Laser-capture microdissection is performed on the Leica LMD laser dissection microscope with motorized stage, phase contrast and epi-fluorescence. The technology allows targeted harvest of fluorescently (or otherwise) labeled single cells or circumscribed brain areas and subsequent isolation of nucleic acids for downstream analyses (qPCR, DNA microarray assessment, etc.).
These are usually applied to specific problems, where the investigator’s laboratory already has basic expertise in a technology but additional expert guidance is needed with some aspect. Core personnel also help with experimental design to maximize the likeliness of a successful outcome. Consultations at a later phase can evolve into collaborative services.