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Reconstruction
Display image description
Maximum intensity projection of a tomographic reconstruction of a selectively stained spiny dendrite from a striatal medium spiny neuron from the neostriatum of a wildtype mouse.
Full resolution image description
a .tar file containing both the mrc .rec and the Analyze version of the reconstructed volume.
Volume_dimension
424, 924, 171
Animation description
A quicktime file of the maximum intensity projection from the reconstructed volume, rotated about the y-axis

Image 2D
Display image description
A 512 by 512 image of a spiny dendrite from the tilt series for a tomographic reconstruction.
Full resolution image description
a .tar file containing the .preali, .ali, .fid and .seed file for an IMOD volume reconstruction.
Animation description
a .mpg file of the tilt series used for tomographic reconstruction

Segmentation
Display image description
Shaft and spines of spiny dendrite specimen extracted by using the object tools in Analyze and then applying a simple threshold
Segmentation file description
a .tar file containing the segmented objects from this spiny dendrite specimen.

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

CCDB:31*
Project: P1207
Project name
Correlative microscopic characterization of dendritic spines in a transgenic mouse model of hyperdopaminergia: The dopamine transporter knockout mouse
Description
Multiscale characterization of DAT KO transgenic mouse
Funding agency
NIH
Leader(s)
Diana Price
Collaborator(s)
Aki Laakso
Michele Cyr
Maryann Martone
Naoko Yamada
Andrea Thor
Monica Berlanga
Start date
01-01-2003
End date
unspecified
 
Experiment
Experiment ID
19
Experiment date
04-22-2003
Title
P1207 Experiment 5
Purpose
EMT reconstructions of medium spiny neuron dendrites
Experimenter(s)
Diana Price
Masako Terada
Andrea Thor
Microscopy product
Microscopy product ID
31
Instrument
Hitachi UHVEM
Microscopy type
UHVEM
Product type
single axis tilt series
Image basename
wt1_g16
Spatial Axis Image Size Pixel Size
X 1024px 0.022 µm
Y 1024px 0.022 µm
Subject
Species
mouse
Scientific name
mus musculus
Strain
C57BL/129SvJ
Group by
genetic manipulation
Treatment
NA
Age
7 months
Age class
adult
Tissue section
Anatomical location
neostriatum
Microtome
vibratome
Tissue product storage
p1207 Slide Box 1
Thickness
100 µm
Specimen description
Organ
brain
System
central nervous
Structure
spiny dendrite
Cell type
medium spiny neuron
Atlas coordinate
0, 0, 0,
Imaging parameters
Type
Light microscopy product
Immersion medium
air
Mounting medium
resin
Lens magnification
X
Numerical aperture
0.5
Specimen preparation
Protocol used
Experiment #5DAT KO mouse 04/22/03Description: Photoconverted dye-filled striatal medium spiny neurons for EMAnimal Info:ID#wt3wt4 Weight: 34g32g DOB: 9/30/029/30/02Protocol1. Perfusion (at Duke) Nembutal; 4% paraformaldehyde + 0.1% gluteraldehyde2. Sectioned on Vibratome (at NCMIR)Thickness = 100 micronsStore in 1X PBS in fridge3.Fill cells with Lucifer yellow4.Store slices with filled cells in 4% para in fridge5.Wash 6x with PBS 1X (on ice)6. When ready to begin photoconversion, turn on the chiller in confocal room. Set at ~4?C. The refrigerator unit should be set at TEMP < 45?C. Switch ON. Stage needs around 20 minutes to come to temperature. Pull unit out into hallway (to avoid increase in temperature).6.Place slices in 2% glut/PBS on ice for 15 minutes0.8 ml 25% gluteraldehyde2 ml 5x PBS6.2ml ddH207.Briefly wash slices in PBS8.Place slices in PBS/glycine for a few minutes38 mg glycine10 ml 1x PBS9.Follow instructions for Photoconversion of Lucifer Yellow-filled cells10. After photoconversion, remove DAB solution and wash slice 3x 10 minutes in generous volumes of PBS on ice. Must remove all DAB before beginning osmification.Microwaving protocol for osmication, dehydration, and embedding of photoconverted slices*Prepare Resin mix and let it sit covered and undisturbed until needed (instructions by fume hood in embedding area).*Rinse slices with a generous amount of cold 1X PBS on ice for ~ 10 min.*Turn on circulating bath (over 20?C, ~ RT): water bath (left hand side) will fill. *Insert temperature probe*Fill other T-beaker with water*Set temperature to 35?C*Open new bottle of 100% ethanol and prepare following dilutions:90% ethanol70% ethanol50% ethanol*Make up osmium solution under fume hood and chill on ice*1% osmium tetroxide in PBS on ice.2.0 ml PBS 5Xthen 5.5 2x distilled H2O2.5 ml Osmium 4%*Rinse w/ 2x distilled H2O ? 3 x 5min*Warm up microwave for 2 minutes on high*Label tubes & place in rack on ice*Fill tubes with osmium solution (w/ meniscus at 0.5)*Using glass hooks, transfer slices to tubes*Remove temperature probe & set temp above 50?C.*Put rack w. tubes in for 40 sec at full power*Change rear water load in T-beaker*Change osmium solution on ice and microwave for another 40 seconds at full power*Rinse samples for 2 minutes in distilled water on benchtop (at RT)*Insert petri bath with H2O under rack*Dehydration steps (2 x 40 seconds per step; all @ 35?C)1st2nd50% EtOH70% EtOH90% EtOH100% EtOH100% Acetone*All of the dehydration steps should be carried out in microcentrifuge tubes filled with 600 ml of solution. Temperature probe should be in petri dish and set for 35. Change water in rear water load when warm to touch.*Change from water to acetone in petri bath under rack ? check acetone bath level every 3 minutes*Infiltration steps (both @ 50?C): With a 50/50 mixture of resin and acetone:1 x 15 min1:1 Resin:acetone* Check rear water load at 7.5 minutesSwitch to 100% resin for 3 x 10 minutes:1st2nd3rd100% Resin*Periodically check rear water load*Flat embed samples between mould release slides and place in embedding oven under vacuum.
Imaging product type
Type
Single tilt
Description
singlet_desc
Min range
-66 degrees
Max range
66 degrees
Tilt increment
2 degrees
Imaging product type
Type
Through focus
Description
transmitted light z series through photoconverted medium spiny neuron
Z step
.25 microns
Psf file
061603B
Notes
subject id: WT2A#2 experiment 5 two injected cells on slide: 061603C and 061603B



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