Triple labeled confocal image of hippocampal area CA1 of a transgenic mouse engineered to overexpress alpha synuclein under the Thy-1 promotor, immunolabeled for mGluR5 (red), alpha synuclein (green) and counterstained with DAPI (blue) to reveal cell nuclei.
Full resolution image description
Zip archive containing the 3 channel image file in tiff format (11200dd_RGB.tif). Also included is the .oif header file generated by the Olympus Fluoview, which gives additional detail on microscope settings.
Localization of Metabotropic Glutamate Receptors in Alpha Synuclein Overexpressing Mouse
Characterization of staining for mGluR5 glutamate receptor in animal model of Parkinsonian disorders
Branfman Family Foundation
Comparison of mGluR5 and synuclein staining
To determine the relationship between mGluR5 and alpha synuclein staining in different lines of alpha synuclein overexpressing mouse
Microscopy product ID
Olympus Fluoview 1000
laser scanning confocal
Overexpression of human alpha synuclein under Thy1 promotor
central nervous system
Light microscopy product
Prolong (Molecular Probes)
Olympus PlanApo 60X oil
DAPI was added to the mounting medium
For more information on transgenic animals, see: Rockenstein et al 2002 E. Rockenstein, M. Mallory, M. Hashimoto, D. Song, C.W. Shults, I. Lang and E. Masliah, Differential neuropathological alterations in transgenic mice expressing alpha-synuclein from the platelet-derived growth factor and Thy-1 promoters, J Neurosci Res 68 (2002), pp. 568 578Specimen processing: Tissue section acquisition from transgenic animalsAnimals were deeply anesthetized with Nembutal" (pentobarbital) and perfused via intracardiac catheterization. Perfusion with oxygenated Ringer's solution containing 250U/ml heparin, 0.2 mg/ml xylocaine and 1% dextrose was followed 4% paraformadehyde in 0.1 M phosphate buffer solution (PBS) (both at 37 degrees Celsius). The brains were carefully removed from the skull and postfixed for 1 hour in the same fixative used in the perfusion. The brain was blocked and cut into 2 mm thick sections using an acrylic brain matrix (David Kopf; Tujunga, CA) to facilitate reproducibility of sections. These thick sections were then sectioned into 80 micron thick coronal sections using a Vibratome (VT1000E, Leica Microsystems, Wetzlar , Germany).Specimen processing: ImmunocytochemistryTissue sections were incubated with monoclonal anti- a-syn (1:250; BD Transduction Laboratories, San Diego, CA, Catalog #AB610787) and rabbit anti-mGluR5 (1:250; Chemicon, Temecula, CA, Catalog #AB5675) followed by incubation with donkey anti-mouse Alexa Fluor 488 (1:100, Molecular Probes, Carlsbad, CA) and donkey anti-rabbit RRX (1:100, Jackson Immunoresearch Laboratories, Inc., West Grove, PA, USA) overnight at 4C. The immunolabeling procedure consisted of the following steps: (1) 6x5 min rinses in 0.1 M PBS; (2) 1 hr blocking step in PBS containing 3% normal donkey (NDS), 0.1% Triton X-100, 1% fish gelatin, and 1% BSA; (3) 48 hr incubation in primary antibodies diluted in working buffer (PBS, 1% NDS) at 20 degrees C; (4) 6 x 5 minute rinsed in working buffer; (5) 24 hr incubation in working buffer containing donkey anti-mouse Alexa Fluor 488 (Molecular Probes, Carlsbad, CA) and donkey anti-rabbit RRX (Jackson Immunoresearch Laboratories, Inc., West Grove, PA, USA). (6) 6 x 10 min rinses in working buffer; (7) 3 x 10 min rinses in PBS; (8) the sections were free floated onto slides and coverslipped using ProLong mounting media (Invitrogen Molecular Probes, Carlsbad, CA) with DAPI nuclear stain. Controls for the mGluR5 antibody experiments included both preabsorption with the control peptide (Chemicon, Catalog #AG374), as well as primary omission studies, which both revealed a lack of non-specific staining. Controls for other antibodies used were performed via omission of primary antibodies, and revealed no non-specific staining. All steps were conducted at 4 degrees C, on wet ice and with ice-cold solutions.
Imaging product type
Only a single optical slice was acquired for each image.
Imaging product type
Survey sections through different brain regions
Diana Price, Edward Rockenstein, Eliezer Masliah, Mark Ellisman CCDB:4059, mus musculus. CIL. Dataset. https://doi.org/doi:10.7295/W9CCDB4059