High-voltage-activated calcium channels are hetero-oligomeric protein complexes that mediate multiple cellular processes, including the influx of extracellular Ca(2+), neurotransmitter release, gene transcription, and synaptic plasticity. These channels consist of a primary α(1) pore-forming subunit, which is associated with an extracellular α(2)δ subunit and an intracellular β auxiliary subunit, which alter the gating properties and trafficking of the calcium channel. The cellular localization of the α(2)δ(3) subunit in the mouse and rat retina is unknown. In this study using RT-PCR, a single band at ∼ 305 bp corresponding to the predicted size of the α(2)δ(3) subunit fragment was found in mouse and rat retina and brain homogenates. Western blotting of rodent retina and brain homogenates showed a single 123-kDa band. Immunohistochemistry with an affinity-purified antibody to the α(2)δ(3) subunit revealed immunoreactive cell bodies in the ganglion cell layer and inner nuclear layer and immunoreactive processes in the inner plexiform layer and the outer plexiform layer. α(2)δ(3) immunoreactivity was localized to multiple cell types, including ganglion, amacrine, and bipolar cells and photoreceptors, but not horizontal cells. The expression of the α(2)δ(3) calcium channel subunit to multiple cell types suggests that this subunit participates widely in Ca-channel-mediated signaling in the retina.
A core facility that provides immunostaining (frozen and paraffin embedded tissues) and image processing services in confocal microscopy, light microscopy and immunostaining for cells and tissues including confocal microscopy, brightfield and fluorescence microscopy, and immunoperoxidase and immunofluorescence staining. First time users must schedule an initial meeting of the P.I., future users and core staff. During this meeting, the project and experimental details will be discussed. Core staff could suggest which microscopy modality works best for the project as well as consultation on strategies to accomplish the imaging task. Users have the option of choosing service work or independent use.
Core facility that provides the following services: Bio-imaging instrumentation training, Bio-Imaging data analysis service. The Bio-Imaging facility currently provides optical and video microscopy services.The facility is equiped with two confocal microscopes, a Leica TCS SP2, and a Perkin Elmer UltraVIEW ERS Spinning Disc confocal . In addtion to confocal microscopy the facility is also equipped for bright field, phase contrast, modulation contrast and epi fluorescence microscopy. The facility has two separate wide field image analysis stations. A Nikon Optiphot microscope equipped with a color video system and Compix imaging software, and a Nikon Eclipse TE200 inverted scope. This inverted scope in specially configured to perform calcium ratio imaging is equipped with Molecular Devices Meta Fluor software The facility has recently expanded with the aquisition of a JEOL JEM-100C/CX Transmission Electron Microscope. The facility also is equiped with Typhoon 9410 scanner for the quantitation and localization of sub cellular fluorescent and radioactive molecules. In addition a Densitometer allows for the analysis of 1 and 2 D gels, Southern, northern and western blots and RFPL gels. The facility is also equipped with color and B&W laser printers for the production of publication quality graphics and presentation materials.
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