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Reconstruction
Display image description
Large scale brain mosaic at the level of the anterior hippocampus showing the localization of alpha synuclein (green) in a normal mouse. Section was counterstained with a nuclear stain (blue) to reveal the locations of cell somata. Full resolution image may be viewed by clicking on the Neuroinformatica link (N) next to the thumbnail.
Full resolution image description
Manual Alignment

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

CCDB:36*  Cite 
Project: P1187
Project name
Correlated Imaging Approaches and Multiscale Databases for Research in Parkinson's Disease
Description
characterization of a mouse model of human alpha synuclein overexpressor
Funding agency
The Branfman Family Foundation
Leader(s)
Diana Price
Collaborator(s)
M.H. Ellisman; M. Martone; G.A. Johnson; E. Masliah
Start date
09-01-2002
End date
09-01-2002
 
Experiment
Experiment ID
21
Experiment date
07-28-2003
Title
Alpha-synuclein immunolabeling for large-scale mapping study
Purpose
to determine the distribution of alpha-synuclein immunolabeling in control and transgenic mouse tissue
Experimenter(s)
Diana Price
Microscopy product
Microscopy product ID
36
Instrument
BioRad RTS 2000MP Multiphoton
Microscopy type
multiphoton
Product type
optical section series/mosaic
Image basename
102203a
Spatial Axis Image Size Pixel Size
X 49px 0.237 µm
Y 59px 0.237 µm
Subject
Species
mouse
Scientific name
mus musculus
Strain
Unspecified
Group by
genetic manipulation
Treatment
None
Age
291 days
Age class
adult
Tissue section
Anatomical location
large scale at level of anterior hippocampus
Microtome
vibratome
Tissue product storage
p1187 #2
Thickness
80 µm
Specimen description
Organ
brain
System
central nervous system
Map location
View location
Imaging parameters
Type
Light microscopy product
Immersion medium
oil
Mounting medium
vectashield with slow fade
Lens
Nikon Plan Fluor
Lens magnification
X
Numerical aperture
1.4
Refractive index
1
Specimen preparation
Protocol used
P1187 Exp. 10A 2-photon Study: Branfman Project 7/28/03Description: alpha-synuclein + nuclear stain for large scale mapping study in striatal regionProtocol1. Perfusion Nembutal; 4% paraformaldehyde 2 hr. postfix in 4% para2. Sectioned on Vibratome Thickness = 80 microns 3 blocks at 2 mm each from anterior (A, B, C) + cerebellum3. sections stored in cryoprotectant at -20 (7/30/03)4. Wash 6x with PBS 1X (on ice)5. Make up blocking buffer PBS w/o NaCl = buffer used Total amount needed = 33 mls x 3 For each 33 ml: Ingredient Amount 0.8 PBS 6.6 ml 5X PBS + 24.2 ml 2x distilled H20 3% NDS ( 24 , 4) 0.96 ml 1% fish gel 0.33 ml 0.1% Triton X-100 0.0332 ml 1% BSA 0.33 g6. Block slices (1 hr) in blocking buffer7. Make up working buffer Use blocking buffer to dilute to working buffer Ingredient 200ml 150ml 100ml 50ml Blocking 20 ml 15 ml 10 ml 5 ml buffer 0.1% Triton 0.2 ml 0.15 ml 0.1 ml 0.05 ml 1X PBS 180 ml 135 ml 90 ml 45 ml8. Wash 1X5 minutes with working buffer9. Dilute 1o Abs in working buffer anti-alpha-synuclein; Host = Rabbit; 1:500 = 4 microliters in 2 ml WB Vial Contents/Tx Total Volume Amt Ab added/vial 1. a-synuclein 2 ml 4 + 2 ?l 2. Control 2 ml Omitted 4 microliters x 2 vials= 8 x 3 animal = 24 microliters total alpha-synuclein10. Place on shaker in cold room labeled & covered with aluminum foil overnight11. Wash 6x with working buffer12. Prepare 2o Abs (for confocal immunolabeling) donkey a.....rabbit AF488 @ 1:100 (MBIRN Box 5)13. Let sit on cold room shaker covered with foil for 2-24 hrs14. Prepare nuclear stain a. Final solution = equal parts 2xPBS + 1:100 Hoescht 33342 in ddH2O b. Prepare each separately. c. Once added together, you should not observe any precipitation. d. If precipitation is observed?. Do not use the solution! e. 2 ml x total number of vials = total ml solution needed15. 30 min staining with nuclear stain16. Wash 6x with 1X PBS 0.8
Imaging product type
Type
Optical section
Z resolution
2.5 um