4051
Diana Price
purpose
Comparison of mGluR5 and synuclein staining
Triple labeled confocal image of hippocampal ara CA3 of a non-transgenic mouse, immunolabeled for mGluR5 (red), alpha synuclein (green) and counterstained with DAPI (blue) to reveal nuclei.
<![CDATA[http://ccdb.ucsd.edu/sand/main?event=rd&type=512r&mpid=4051]]>
Zip archive containing the 3 channel image file in tiff format (112006bb_RGB.tiff). Also included are the .oif header file generated by the Olympus Fluoview, which give additional detail on microscope settings.
/telescience/home/CCDB_DATA_USER.portal/P1723/Experiment_3482/Subject_253/Tissue_366/Microscopy_4051/112006bbb_img.zip
oil
Olympus PlanApo 60X oil
X
Prolong (Molecular Probes)
DAPI was added to the mounting medium
1.42
Only a single optical section was taken for each image.
survey sections of different regions of brain
2006-11-20 00:00:00.0
4051
112006bbb
Olympus Fluoview 1000
laser scanning confocal
SURVEY
.207 um/pixels
1024
.207 um/pixels
1024
Edward Rockenstein
Eliezer Masliah
Mark Ellisman
Branfman Family Foundation
Diana Price
Localization of Metabotropic Glutamate Receptors in Alpha Synuclein Overexpressing Mouse
P1723
brain
1
central nervous system
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.
triple label
Genetic Modification
adult
unspecified
Eliezer Masliah
mus musculus
mouse
C57BL/6-DBA/2
Vibratome
80 um
None