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Image 2D
Display image description
Triple labeled confocal image of the cerebellum of a non-transgenic mouse, immunolabeled for mGluR5 (red), alpha synuclein (green) and counterstained with DAPI (blue) to reveal nuclei. Staining of the granule cell layer appears to be uneven, likely due to unevenness in the section.
Full resolution image description
Zip archive containing the 3 channel image file in tiff format (112006bbbbb_RGB.tiff). Also included is the .oif header file generated by the Olympus Fluoview, which gives additional detail on microscope settings.

License
Attribution Only: This image is licensed under a Creative Commons Attribution License. View License Deed | View Legal Code

CCDB:4053*
Project: P1723
Project name
Localization of Metabotropic Glutamate Receptors in Alpha Synuclein Overexpressing Mouse
Description
Characterization of staining for mGluR5 glutamate receptor in animal model of Parkinsonian disorders
Funding agency
Branfman Family Foundation
Leader(s)
Diana Price
Collaborator(s)
Edward Rockenstein
Eliezer Masliah
Mark Ellisman
Start date
unspecified
End date
unspecified
 
Experiment
Experiment ID
3482
Title
Comparison of mGluR5 and synuclein staining
Purpose
To determine the relationship between mGluR5 and alpha synuclein staining in different lines of alpha synuclein overexpressing mouse
Experimenter(s)
Diana Price
Microscopy product
Microscopy product ID
4053
Instrument
Olympus Fluoview 1000
Microscopy type
laser scanning confocal
Product type
SURVEY
Image basename
112006bbbbb
Spatial Axis Image Size Pixel Size
X 1024px 0.207 um/pixels
Y 1024px 0.207 um/pixels
Subject
Species
mouse
Scientific name
mus musculus
Strain
C57BL/6-DBA/2
Group by
Genetic Modification
Treatment
None
Age class
adult
Tissue section
Microtome
Vibratome
Thickness
80 µm
Specimen description
Organ
brain
System
central nervous system
Imaging parameters
Type
Light microscopy product
Immersion medium
oil
Mounting medium
Prolong (Molecular Probes)
Lens
Olympus PlanApo 60X oil
Lens magnification
X
Numerical aperture
1.42
Notes
DAPI was added to the mounting medium
Specimen preparation
Protocol used
Specimen 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: Immunocytochemistry Tissue 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
Type
Optical section
Description
Only a single optical section was taken for each image.
Imaging product type
Type
Survey
Description
survey sections of different regions of brain




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