Application of Polyethylene Glycol (PEG) Embedding and Rotary-Replication Techniques to the Study of Photoreceptor Synaptic Ribbon Ultrastructure

1988 ◽  
Vol 46 ◽  
pp. 268-269
Author(s):  
D. Howard Dickson ◽  
Stephan C. WhitefieLd ◽  
Christine A. Morrison
Keyword(s):  
Synaptic Vesicles ◽  
Freeze Substitution ◽  
Major Advance ◽  
Ice Crystal ◽  
Synaptic Ribbon ◽  
Sodium Cacodylate ◽  
Embedding Technique ◽  
Intimate Association ◽  
Cacodylate Buffer ◽  
The Cost

Synaptic ribbons (SRs) are electron-dense, LameLLar specializations found in the retina, inner ear and pineal. Although it has been suggested that SRs may have a functional role in the maintenance of synaptic terminal shape or in orienting synaptic vesicles to the presynaptic membrane neither hes been confirmed. Their intimate association with synaptic vesicles has however supported the Latter hypothesis. Controversy also exists with respect to diurnal rhythmicity of these organelles. Recently, a major advance in our knowledge of SR structure was achieved through the application of freeze-slamming and freeze-substitution techniques. However, the cost and complexity of these techniques, together with the Limitations imposed by freezing rate and ice-crystal damage has Led us to explore the use of the PEG-embedding technique to further our study of SRs in retina and pineal.Chick retinas were prepared following the technique of Kondo. Tissues were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer containing 0.5% tannic acid at pH 7.4 for 2 hr, washed in buffer and postfixed in 1% OsO4 for 1 hr.

scholarly journals AN ELECTRON MICROSCOPIC CHARACTERIZATION OF CLASSES OF SYNAPTIC VESICLES BY MEANS OF CONTROLLED ALDEHYDE FIXATION

1970 ◽  
Vol 44 (1) ◽  
pp. 115-124 ◽  
Author(s):  
David Bodian
Keyword(s):  
Spinal Cord ◽  
Synaptic Vesicles ◽  
Osmium Tetroxide ◽  
Sodium Cacodylate ◽  
Electron Microscopic ◽  
Osmium Tetroxide Solution ◽  
Aldehyde Fixation ◽  
Cacodylate Buffer ◽  
Peripheral Axon

Examination of variables of aldehyde fixation that may affect the shape of agranular synaptic vesicles has revealed that even brief storage of aldehyde-perfused nervous tissue pieces in cacodylate buffer, prior to hardening in osmium tetroxide, has an unusually severe flattening effect on agranular vesicles of a particular type. These are the vesicles of peripheral cholinergic axon endings, and of certain central synaptic bulbs. Types of synaptic bulbs can now be further defined on the basis of shape of agranular synaptic vesicles under controlled conditions of aldehyde fixation. Previously described "S" bulbs in the spinal cord contain uniformly spheroid vesicles, which are wholly resistant to flattening. Previously described "F" bulbs contain somewhat smaller agranular vesicles that are flattened after aldehyde fixation, even when this is followed by prompt hardening in osmium tetroxide solution. A third type, previously characterized as having irregularly round agranular vesicles after the above treatment, contains only severely flattened vesicles when the osmium tetroxide hardening is preceded by even a brief wash with sodium cacodylate buffer containing sucrose. Moreover, the "third" type is characteristic of all cholinergic peripheral axon endings examined, as well as the large axosomatic ("L") synaptic bulbs of the spinal cord.

High-pressure freezing of cells in suspension for low-voltage scanning electron microscopy (LVSEM)

1991 ◽  
Vol 49 ◽  
pp. 64-65
Author(s):  
Marek Malecki ◽  
James Pawley ◽  
Hans Ris
Keyword(s):  
Electron Microscopy ◽  
High Pressure ◽  
Low Voltage ◽  
Culture Media ◽  
Cell Structure ◽  
Freeze Substitution ◽  
Ice Crystal ◽  
High Pressure Freezing ◽  
Cell Culture Media ◽  
Voltage Scanning

The ultrastructure of cells suspended in physiological fluids or cell culture media can only be studied if the living processes are stopped while the cells remain in suspension. Attachment of living cells to carrier surfaces to facilitate further processing for electron microscopy produces a rapid reorganization of cell structure eradicating most traces of the structures present when the cells were in suspension. The structure of cells in suspension can be immobilized by either chemical fixation or, much faster, by rapid freezing (cryo-immobilization). The fixation speed is particularly important in studies of cell surface reorganization over time. High pressure freezing provides conditions where specimens up to 500μm thick can be frozen in milliseconds without ice crystal damage. This volume is sufficient for cells to remain in suspension until frozen. However, special procedures are needed to assure that the unattached cells are not lost during subsequent processing for LVSEM or HVEM using freeze-substitution or freeze drying. We recently developed such a procedure.

Oligodendrocytes in Developing Hameter Cerebral Cortex

1976 ◽  
Vol 34 ◽  
pp. 126-127
Author(s):  
MB. Tank Buschmann
Keyword(s):  
Cerebral Cortex ◽  
Optic Nerve ◽  
Frontal Cortex ◽  
Osmium Tetroxide ◽  
Sodium Cacodylate Buffer ◽  
Sodium Cacodylate ◽  
Tissue Samples ◽  
Cacodylate Buffer ◽  
Layer V ◽  
Adult Hamster

Development of oligodendrocytes in rat corpus callosum was described as a sequential change in cytoplasmic density which progressed from light to medium to dark (1). In rat optic nerve, changes in cytoplasmic density were not observed, but significant changes in morphology occurred just prior to and during myelination (2). In our study, the ultrastructural development of oligodendrocytes was studied in newborn, 5-, 10-, 15-, 20-day and adult frontal cortex of the golden hamster (Mesocricetus auratus).Young and adult hamster brains were perfused with paraformaldehyde-glutaraldehyde in sodium cacodylate buffer at pH 7.3 according to the method of Peters (3). Tissue samples of layer V of the frontal cortex were post-fixed in 2% osmium tetroxide, dehydrated in acetone and embedded in Epon-Araldite resin.

High-pressure freezing and freeze substitution of plant tissue

1992 ◽  
Vol 50 (1) ◽  
pp. 748-749
Author(s):  
William P. Sharp ◽  
Robert W. Roberson
Keyword(s):  
High Pressure ◽  
Cell Structure ◽  
Leaf Tissue ◽  
Freeze Substitution ◽  
Crystal Formation ◽  
Ice Crystal ◽  
High Pressure Freezing ◽  
Cell Components ◽  
Cold Metal ◽  
Brome Grass

The aim of ultrastructural investigation is to analyze cell architecture and relate a functional role(s) to cell components. It is known that aqueous chemical fixation requires seconds to minutes to penetrate and stabilize cell structure which may result in structural artifacts. The use of ultralow temperatures to fix and prepare specimens, however, leads to a much improved preservation of the cell’s living state. A critical limitation of conventional cryofixation methods (i.e., propane-jet freezing, cold-metal slamming, plunge-freezing) is that only a 10 to 40 μm thick surface layer of cells can be frozen without distorting ice crystal formation. This problem can be allayed by freezing samples under about 2100 bar of hydrostatic pressure which suppresses the formation of ice nuclei and their rate of growth. Thus, 0.6 mm thick samples with a total volume of 1 mm3 can be frozen without ice crystal damage. The purpose of this study is to describe the cellular details and identify potential artifacts in root tissue of barley (Hordeum vulgari L.) and leaf tissue of brome grass (Bromus mollis L.) fixed and prepared by high-pressure freezing (HPF) and freeze substitution (FS) techniques.

Opening images of synaptic vesicles and calcium localization by means of microwave fixation method

1990 ◽  
Vol 48 (2) ◽  
pp. 182-183
Author(s):  
Vinci Mizuhira ◽  
Hiroshi Hasegawa
Keyword(s):  
Synaptic Vesicles ◽  
Room Temperature ◽  
Osmium Tetroxide ◽  
Sampling Procedure ◽  
Fixation Method ◽  
Potassium Oxalate ◽  
X Ray ◽  
Cacodylate Buffer ◽  
Ultrathin Sections ◽  
Microwave Fixation

Microwave irradiation (MWI) was applied to 0.3 to 1 cm3 blocks of rat central nervous system at 2.45 GHz/500W for about 20 sec in a fixative, at room temperature. Fixative composed of 2% paraformaldehyde, 0.5% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.4, also contained 2 mM of CaCl2 , 1 mM of MgCl2, and 0.1% of tannic acid for conventional observation; and fuether 30-90 mM of potassium oxalate containing fixative was applied for the detection of calcium ion localization in cells. Tissue blocks were left in the same fixative for 30 to 180 min after MWI at room temperature, then proceeded to the sampling procedure, after postfixed with osmium tetroxide, embedded in Epon. Ultrathin sections were double stained with an useal manner. Oxalate treated sections were devided in two, stained and unstained one. The later oxalate treated unstained sections were analyzed with electron probe X-ray microanalyzer, the EDAX-PU-9800, at 40 KV accelerating voltage for 100 to 200 sec with point or selected area analyzing methods.

Structural integrity after cold storage of human epidermal model in hypothermosol: A correlative ultrastructural and fluorescent assay

1994 ◽  
Vol 52 ◽  
pp. 270-271
Author(s):  
Henry H. Eichelberger ◽  
John G. Baust ◽  
Robert G. Van Buskirk
Keyword(s):  
Cold Storage ◽  
Structural Integrity ◽  
Culture Media ◽  
Cell Structure ◽  
Natural State ◽  
Sodium Cacodylate ◽  
Ruthenium Tetroxide ◽  
Cacodylate Buffer ◽  
Before And After

For research in cell differentiation and in vitro toxicology it is essential to provide a natural state of cell structure as a benchmark for interpreting results. Hypothermosol (Cryomedical Sciences, Rockville, MD) has proven useful in insuring the viability of synthetic human epidermis during cold-storage and in maintaining the epidermis’ ability to continue to differentiate following warming.Human epidermal equivalent, EpiDerm (MatTek Corporation, Ashland, MA) consisting of fully differentiated stratified human epidermal cells were grown on a microporous membrane. EpiDerm samples were fixed before and after cold-storage (4°C) for 5 days in Hypothermosol or skin culture media (MatTek Corporation) and allowed to recover for 7 days at 37°C. EpiDerm samples were fixed 1 hour in 2.5% glutaraldehyde in sodium cacodylate buffer (pH 7.2). A secondary fixation with 0.2% ruthenium tetroxide (Polysciences, Inc., Warrington, PA) in sodium cacodylate was carried out for 3 hours at 4°C. Other samples were similarly fixed, but with 1% Osmium tetroxide in place of ruthenium tetroxide. Samples were dehydrated through a graded acetone series, infiltrated with Spurrs resin (Polysciences Inc.) and polymerized at 70°C.

scholarly journals Nanoscale dynamics of synaptic vesicle trafficking and fusion at the presynaptic active zone

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Thirumalini Vaithianathan ◽  
Diane Henry ◽  
Wendy Akmentin ◽  
Gary Matthews
Keyword(s):  
Active Zone ◽  
Synaptic Vesicles ◽  
Super Resolution ◽  
Visual Signals ◽  
Dual Function ◽  
Macromolecular Complex ◽  
Synaptic Ribbon ◽  
Modes Of Transmission ◽  
Resolution Imaging ◽  
Specialized Structure

The cytomatrix at the active zone (CAZ) is a macromolecular complex that facilitates the supply of release-ready synaptic vesicles to support neurotransmitter release at synapses. To reveal the dynamics of this supply process in living synapses, we used super-resolution imaging to track single vesicles at voltage-clamped presynaptic terminals of retinal bipolar neurons, whose CAZ contains a specialized structure—the synaptic ribbon—that supports both fast, transient and slow, sustained modes of transmission. We find that the synaptic ribbon serves a dual function as a conduit for diffusion of synaptic vesicles and a platform for vesicles to fuse distal to the plasma membrane itself, via compound fusion. The combination of these functions allows the ribbon-type CAZ to achieve the continuous transmitter release required by synapses of neurons that carry tonic, graded visual signals in the retina.

scholarly journals Sodium cacodylate as antimitotic agent

2014 ◽  
Vol 57 (3) ◽  
pp. 329-340
Author(s):  
Jadwiga A. Tarkowska
Keyword(s):  
Allium Cepa ◽  
Mitotic Spindle ◽  
Sodium Cacodylate ◽  
Antimitotic Agent ◽  
Ultrastructural Studies ◽  
Meristematic Cells ◽  
Cacodylate Buffer ◽  
Root Meristematic Cells ◽  
Dividing Cells

The effect of pure sodium cacodylate on dividing cells was studied. The root meristematic cells of <em>Allium cepa</em> L. (the roots were squashed in acetoorcein) and endosperm cells of <em>Haemanthus katherinae</em> Bak. (<em>in vitro</em> observations) were used. Serious disturbances in karyokinesis and cytokinesis were found that led most often to the formation of polyploid or multinucleate (<em>A. cepa</em>) cells. These results point to damage of the mitotic spindle and phragmoplast. Careful use of cacodylate buffer in ultrastructural studies of microtubules is advised.

scholarly journals FLORAL INITIATION IN THE LONG-DAY PLANT Rudbeckia hirta

HortScience ◽  
1991 ◽  
Vol 26 (6) ◽  
pp. 719A-719
Author(s):  
Richard L. Harkess ◽  
Robert E. Lyons
Keyword(s):  
Methyl Green ◽  
Floral Initiation ◽  
Serial Sections ◽  
Sodium Cacodylate ◽  
Rudbeckia Hirta ◽  
Long Day ◽  
Cacodylate Buffer ◽  
Ethanol Series ◽  
Inductive Photoperiod ◽  
Short Days

A study was undertaken to determine the rate of floral initiation in Rudbeckia hirta. R. hirta plants were grown to maturity, 14-16 leaves, under short days (SD). Paired controls were established by placing half of the plants under long days (LD) with the remainder left under SD. Beginning at the start of LD (day 0), five plants were harvested daily from each photoperiod group for twenty days. Harvested meristems were fixed in 2% paraformaldehyde - 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.0) for 24 hrs, dehydrated in an ethanol series, embedded in paraffin and sectioned at 8 μm. Serial sections were stained with Methyl-green Pyronin, with adjacent sections treated with RNase for nucleic acid comparison. All events of floral initiation were identified, The results of limited inductive photoperiod indicate that 16-18 LD were required for flowering.

scholarly journals Are the Triptans for Migraine Therapy Worth the Cost?

2000 ◽  
Vol 27 (2) ◽  
pp. 111-115 ◽  
Author(s):  
W.J. Becker
Keyword(s):  
Quality Of Life ◽  
Significant Proportion ◽  
Cost Benefit ◽  
New Drugs ◽  
Major Advance ◽  
Improve Quality ◽  
Migraine Therapy ◽  
The Cost ◽  
Severe Migraine

ABSTRACT:The triptans represent a major advance in migraine therapy but their cost per dose greatly exceeds that of many older treatments. There is evidence that for a significant proportion of migraine patients these new drugs can show a positive cost benefit and also improve quality of life. Cost benefit would be expected to be greatest in patients with more severe migraine attacks.

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