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Reconstruction
Display image description
single slice through the middle of a tomographic reconstruction of Purkinje cell spines labeled for F-actin
Full resolution image description
Reconstruction of cerebellar neuropil using phalloidin-photooxidation
Volume_dimension
836, 798, 166
Volume scale
0.0012, 0.0012, 0.0012
Animation description
Slices through cerebellar molecular layer neuropil showing several examples of dendritic spines labeled for F-actin using phalloidin-photooxidation

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

CCDB:22*
Project: P1134
Project name
Actin in dendritic spines
Description
Actin localization in spines of CNS using eosin phalloidin photooxidation
Funding agency
NIH
Leader(s)
Francisco Capani
Collaborator(s)
M Martone; M Ellisman
Start date
01-01-1998
End date
01-01-1998
 
Experiment
Experiment ID
14
Experiment date
10-01-1999
Title
Tomographic reconstruction of phalloidin labeling in cerebellar cortex
Purpose
The subcellular distribution of F-actin in cerebellar Purkinje cell spines
Experimenter(s)
Francisco Capani
Microscopy product
Microscopy product ID
22
Instrument
JEOL4000 IVEM
Microscopy type
IVEM
Product type
single tilt
Image basename
cere14
Spatial Axis Image Size Pixel Size
X 1024px 0.0012 µm
Y 1024px 0.0012 µm
Subject
Species
rat
Scientific name
rattus norvegicus
Strain
Sprague Dawley
Treatment
none
Age class
adult
Tissue section
Anatomical location
cerebellum
Microtome
ultramicrotome
Thickness
1 µm
Specimen description
Organ
brain
System
central nervous system
Structure
neuropil
Cell type
Purkinje neuron
Imaging parameters
Type
Electron microscopy product
Recording medium
film
Magification
40000
Accelerating voltage
400 KeV
Specimen preparation
Protocol used
1) Tissue: Five male Sprague Dawley adult rats were used in this study. The committee on animal studies of UCSD following the NIH guidelines approved all of experimental procedures. Briefly, an intracardiac perfusion was performed under deep anesthesia (containing 50 mg/kg ketamine, 1mg/kg rhompun and 5 mg/kg acetopromazine in sterile saline) with normal rat Ringer's at 35C followed by fixative. For light microscopic analyses, rats were perfused with 4% formaldehyde (made fresh from paraformaldehyde) in cacodylate buffer, pH 7.2. The brain was removed and fixed an additional 2 hr in the same solution at 4C. For electron microscopic studies, a range of fixative strengths were employed containing 2 or 4 % formaldehyde and 0.5%-2.5% glutaraldehyde. The tissue was postfixed for 2 hr in the same fixative. After removal of the brain from the skull, coronal or sagital sections through striatum, cerebellum and hippocampus were cut at a thickness of 50-80 um with a Vibrating Microtome (Leica, model VT 1000E). As a control, we also labeled cultured bovine aortic endothelial cells, fixed using the same conditions as above, which possess characteristic bundles of actin filaments called stress fibers. Details about culturing methods are given in Deerinck et al. (1994).2) Electron microscopic analysis using photooxidation of eosin-phalloidin: Vibratome sections were washed with 50 mM glycine-PBS containing 0.5% cold water fish gelatin to block nonspecific binding. Following 30 min of washing, the sections were incubated with agitation in a solution of 0.05% of eosin-phalloidin in 0.5% cold water fish gelatin/50mM glycine-PBS 2 hr. For light microscopy studies, phalloidin conjugated to rhodamine was also used because of its superior fluorescent quantum yield. As a negative control, the eosin-phalloidin was omitted. Fluorescent and transmitted light images were recorded using a Zeiss Axiovert inverted microscope with a laser scanning confocal attachment (MRC-1024; Bio-RAD Laboratories, Cambridge, MA) and a krypton/argon mixed gas laser. Images were collected digitally using either a 40X oil (n.a. =1.3) or 63X (n.a. =1.4) oil objective and transferred to a graphics program (Adobe Photoshop 5.0).3) Photooxidation: After additional washes in sodium cacodylate buffer, tissue sections labeled with eosin-phalloidin were mounted on glass-welled tissue culture dishes (Mat Tek Corp) pretreated with Cell Tak adhesive (Collaborative Research Inc). Slices were fixed again for 2-5 min with 2% glutaraldehyde in 0.1 M cacodylate buffer, rinsed in buffer for several minutes, and placed in 50mM glycine and potassium cyanide in cacodylate buffer for an additional 5 min to reduce nonspecific staining. Photooxidation was performed on the Zeiss Axiovert described above, equipped with a 75W xenon arc light source. Specimens were viewed with a 40X oil objective, n.a. 1.3. Three areas were chosen for electron microscopic analysis: cerebellar molecular layer, dorsal striatum and hippocampal area CA1. The appropriate areas were located with transmitted light and the pattern of fluorescent labeling was recorded using the confocal attachment at a low laser power setting. The samples were immersed for ten minutes in a solution of 2.8 mM DAB in 0.1 M sodium cacodylate at 4C bubbled with pure O2, final pH 7.4, and then irradiated under conventional epifluorescence using a xenon lamp. The DAB solution was changed every few minutes while the reaction proceeded. Continuous observations were made during the photooxidation procedure using transmitted light. After 6-8 min., a brownish reaction product began to appear in place of the fluorescence. The process was stopped by halting the excitation (Deerinck et al., 1994 ). Following photooxidation, tissue sections were rinsed in 0.1M sodium cacodylate several times and incubated for 30 min with 1% osmium tetroxide in 0.1M sodium cacodylate, pH 7.4. Some sections were fixed for 1 hr in 2.25% glutaraldehyde with 0.2% tannic acid added both in cacodylate buffer. Osmication was done with 0.75 % OsO4 in cacodylate buffer, pH 6, for 1 hr on ice. Treatment with tannic acid and osmication at low pH is known to protect actin filaments from depolymerization during osmium fixation (Pollard and Maupin, 1982). After several washes with ddH20, slices were dehydrated in an ascending ethanol series, infiltrated with Durcopan ACM resin and polymerized for 24 hr at 60C. Thin sections (80-100 nm) and thick sections (0.5-1um) were cut with Reichert Ultracut E using glass knives. Thin sections were examined using a JEOL 100CX electron microscope at 80-100 keV and thick sections were observed using a JEOL JEM-4000EX intermediate voltage microscope (IVEM) at 400 keV. One set of thin sections was poststained with a combination of uranyl acetate and lead citrate, but most were examined without additional counterstain. Stereopairs were generated by tilting the specimen 5 degrees between micrographs.
Imaging product type
Type
Single tilt
Description
singlet_desc
Min range
-60 degrees
Max range
60 degrees
Tilt increment
2 degrees


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