scholarly journals Changes in Extracellular Space of the Mouse Cerebral Cortex During Hydroxyadipaldehyde Fixation and Osmium Tetroxide Post-Fixation

1969 ◽  
Vol 4 (2) ◽  
pp. 437-453
Author(s):  
A. VAN HARREVELD ◽  
F. I. KHATTAB
Keyword(s):  
Cerebral Cortex ◽  
Extracellular Space ◽  
Osmium Tetroxide ◽  
Freeze Substitution ◽  
Fluid Distribution ◽  
Fixed Tissue ◽  
Tissue Conductivity ◽  
Mouse Cerebral Cortex ◽  
Cacodylate Buffer ◽  
Dendritic Structures

Perfusion of the cerebral cortex of mice with a 4.5 and 12.5% hydroxyadipaldehyde (HAA) solution in a cacodylate buffer caused a biphasic change in the tissue conductivity. After a latency of a fraction of a minute the cortical conductivity dropped markedly, reaching a minimum in 1.5-2 min. Then the conductivity increased again. Electron micrographs (EMs) of material perfused with HAA for 15-20 min and post-fixed with osmium tetroxide showed electron-transparent swollen structures, some of which could be identified as dendritic. The extracellular space consisted of 100-200 Å slits between the tissue elements and larger spaces in bundles of small profiles (unmyelinated axons). Cortex frozen after 2 min perfusion with HAA and subjected to substitution in acetone containing 2 % OsO4 at -85 °C showed swollen (dendritic) structures and a paucity of extracellular material in accordance with the conductivity drop. Often tight junctions between the tissue elements were present. Tissue frozen after 15-20 min of HAA perfusion when the conductivity had increased again yielded EMs which were characterized by an abundance of extracellular space between the small profiles. The mitochondria in the swollen (dendritic) structures were enormously enlarged. Cortex perfused for 15-20 min with HAA, post-fixed with OsO4 and then freeze substituted produced EMs resembling those of tissue fixed in the same way but not subjected to freeze substitution. The examination of the fixation process by freeze substitution demonstrated a sequence of major changes in the fluid distribution of the tissue which precludes any direct relationship between the spaces in the normal and fixed tissue.

scholarly journals Perfusion Fixation With Glutaraldehyde and Post-Fixation With Osmium Tetroxide for Electron Microscopy

1968 ◽  
Vol 3 (4) ◽  
pp. 579-594
Author(s):  
A. VAN HARREVELD ◽  
F. I. KHATTAB
Keyword(s):  
Cerebral Cortex ◽  
Extracellular Space ◽  
Water Distribution ◽  
Osmium Tetroxide ◽  
Circulatory Arrest ◽  
Freeze Substitution ◽  
Tissue Conductivity ◽  
Initial Drop ◽  
Previously Treated ◽  
Perfusion Fixation

The conductivity of cerebral cortex drops during perfusion with glutaraldehyde in 5 min to about 60% of the original value, to remain unchanged during the subsequent 10-15 min of perfusion. Circulatory arrest causes a similar drop in the tissue conductivity. Perfusion of asphyxiated tissue with glutaraldehyde does not produce additional major changes in the conductivity. Perfusion of the cortex with an osmium tetroxide solution causes an initial drop in conductivity. However, after about 3 min this trend is reversed and the conductivity increases again to close to the pre-perfusion value. Perfusion of asphyxiated cortex with OsO4 causes a marked increase of the conductivity. So does perfusion with an OsO4 solution of tissue previously treated with glutaraldehyde. One interpretation of these impedance changes is that glutaraldehyde perfusion causes, like asphyxiation, a transport of extracellular material into the intracellular compartment and that during OsO4 perfusion an extracellular space is again created. This possibility is supported by electron micrographs made of this material. Cerebral cortex perfused with glutaraldehyde and post-fixed with OsO4 shows electron-transparent dendritic elements and to a lesser extent pre-synaptic terminals, which seem to be swollen. When the cortex is flooded with a salt solution during glutaraldehyde perfusion the dendrites exhibit ballooning in the surface layer of the cortex, suggesting that the fluid on the cortex participates in the swelling. The tissue elements in the glutaraldehyde-perfused and OsO4 post-fixed cortex are separated by narrow extracellular spaces. The latter may have been produced by the OsO4 perfusion as is suggested by a comparison of micrographs prepared by freeze substitution (which tends to preserve the water distribution) of glutaraldehyde-perfused but not post-fixed cortex with micrographs of cortex treated with OsO4 after the glutaraldehyde perfusion. In accordance with the conductivity changes, the former micrographs showed very little extracellular space, and in many places tight junctions, whereas the latter showed clefts between the tissue elements.

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.

Microvasculature and structure of hemal lymph nodes in the wall of the rabbit bladder: a vascular corrosion casting and EM study

1995 ◽  
Vol 53 ◽  
pp. 1074-1075
Author(s):  
F.E. Hossler ◽  
M.I. McKamey ◽  
F.C. Monson
Keyword(s):  
Lymph Nodes ◽  
Light Microscopy ◽  
Corrosion Casting ◽  
Fixed Tissue ◽  
Infusion Pressure ◽  
India Ink ◽  
Cacodylate Buffer ◽  
Rabbit Bladder ◽  
Lateral Walls ◽  
Comprehensive Study

A comprehensive study of the microvasculature of the normal rabbit bladder, revealed unusual "capillary glomeruli" along the lateral walls. Here they are characterized as hemal lymph nodes using light microscopy, SEM, TEM, ink injection, and vascular casting.Bladders were perfused via a cannula placed in the abdominal aorta with either 2% glutaraldehyde in 0.1M cacodylate buffer (pH 7.4) for fixation, 10% India ink in 0.9% saline and 0.1M phosphate (pH 7.4) for vessel tracing, or resin (Mercoximethylmethacrylate: catalyst, 4:1:0.3; Ladd Research Industries) for vascular corrosion casting. Infusion pressure was 100mm Hg. Fixed tissue was sectioned from epon-araldyte resin, and stained with toluidine blue for light microscopy, and lead and uranium for TEM. Ink injected tissue was photographed directly from saline-filled bladders illuminated from below. Resin-filled tissue was macerated in 5% KOH and distilled water. Casts were critical point dried, sputter coated with goldpalladium, and examined by routine SEM at 10 KV.

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.

TEM comparison of osmium vs osmium with potassium ferricyanide secondary fixatives and the impact of secondary fixative temperature on tissue preservation/contrast quality

1996 ◽  
Vol 54 ◽  
pp. 910-911
Author(s):  
J. W. Horn ◽  
B. J. Dovey-Hartman ◽  
V. P. Meador
Keyword(s):  
Osmium Tetroxide ◽  
Potassium Ferricyanide ◽  
Electron Microscopic ◽  
Transmission Electron ◽  
Microscopic Evaluation ◽  
Cacodylate Buffer ◽  
Transmission Electron Microscopic ◽  
Glutaraldehyde Solution ◽  
Temperature Impact ◽  
The Impact

Osmium tetroxide (OsO4) is a universally used secondary fixative for routine transmission electron microscopic evaluation of biological specimens. Use of OsO4 results in good ultrastructural preservation and electron density but several factors, such as concentration, length of exposure, and temperature, impact overall results. Potassium ferricyanide, an additive used primarily in combination with OsO4, has mainly been used to enhance the contrast of lipids, glycogen, cell membranes, and membranous organelles. The purpose of this project was to compare the secondary fixative solutions, OsO4 vs. OsO4 with potassium ferricyanide, and secondary fixative temperature for determining which combination gives optimal ultrastructural fixation and enhanced organelle staining/contrast.Fresh rat liver samples were diced to ∼1 mm3 blocks, placed into porous processing capsules/baskets, preserved in buffered 2% formaldehyde/2.5% glutaraldehyde solution, and rinsed with 0.12 M cacodylate buffer (pH 7.2). Tissue processing capsules were separated (3 capsules/secondary fixative.solution) and secondarily fixed (table) for 90 minutes. Tissues were buffer rinsed, dehydrated with ascending concentrations of ethanol solutions, infiltrated, and embedded in epoxy resin.

Immunocytochemistry after fast freeze fixation-freeze substitution: Advantages and limits

1990 ◽  
Vol 48 (3) ◽  
pp. 42-43
Author(s):  
Marie-Thérèse Nicolas
Keyword(s):  
Osmium Tetroxide ◽  
Freeze Substitution ◽  
Acrylic Resin ◽  
Expected Improvement ◽  
List Type ◽  
Chemical Fixation ◽  
Freeze Fixation ◽  
Liquid Chemical ◽  
Luciferase Enzyme ◽  
Lower Background

An alternative to aqueous chemical fixation consists in immobilizing physically the specimen by freezing it as fast as possible without using any cryoprotectant. This Fast Freeze Fixation (FFF) followed by Freeze Substitution (FS) avoids osmotic artefacts due to the slow penetration of liquid chemical fixative. Associated with Immuno-Gold labeling (IGS), FFF-FS allows a more precise localization of antigens.Using the bioluminescent bacteria Vibrio harveyi, a comparison of IGS with an antibody directed against its luciferase (enzyme of the luminescent reaction) has been done after liquid chemical fixation versus FFFFS. This later technique, beside an expected improvement of the ultrastructure always shows a better preservation of antigenicity and a lower background. In the case of FFF-FS technique (Figure 3):–labeling in acrylic resin (LRWhite) is 2 to 4 fold more intense than in epoxy resin (Epon),–but the ultrastructure is always better in Epon.–but the ultrastructure is always better in Epon.–The addition of fixatives in the substitution medium, results in a decrease of labeling which is more important in the case of a mixture of fixatives than with osmium tetroxide alone; with one exception: the substitution with glutaraldehyde which produces a dramatic increase in the density of the labeling but also, at the same time, a swelling of the cells of about 30%.

Ultrastructural and immunocytochemical studies of the rat hypothalamo-neurohypophysial system processed by rapid freezing and freeze-substitution fixation

1990 ◽  
Vol 48 (3) ◽  
pp. 884-885
Author(s):  
Seiji Shioda ◽  
Yasumitsu Nakai ◽  
Atsushi Ichikawa ◽  
Hidehiko Ochiai ◽  
Nobuko Naito
Keyword(s):  
Osmium Tetroxide ◽  
Freeze Substitution ◽  
Ultrastructural Localization ◽  
Wistar Rat ◽  
Neurosecretory Cells ◽  
Rapid Freezing ◽  
Sodium Metaperiodate ◽  
Tissue Sections ◽  
Ultrathin Sections ◽  
Electron Microscopes

The ultrastructure of neurosecretory cells and glia cells in the supraoptic nucleus (SON) of the hypothalamus and the neurohypophysis (PN) was studied after rapid freezing followed by substituion fixation. Also, the ultrastructural localization of vasopressin (VP) or its carrier protein neurophys in II (NPII) in the SON and PN was demonstrated by using a post-embedding immunoco1loidal gold staining method on the tissue sections processed by rapid freezing and freeze-substitution fixation.Adult male Wistar rat hypothalamus and pituitary gland were quenched by smashing against a copper block surface precooled with liquid helium and freeze-substituted in 3% osmium tetroxide-acetone solutions kept at -80°C for 36-48h. After substituion fixation, the tissue blocks were warmed up to room temperature, washed in acetone and then embedded in an Epon-Araldite mixture. Ultrathin sections mounted on 200 mesh nickel grids were immersed in saturated sodium metaperiodate and then incubated in each of the following solutions: 1 % egg albumin in phosphate buffer, VP or NPII (1/1000-1/5000) antiserum 24h at 4°C, 3) colloidal gold solution (1/20) 1h at 20°C. The sections were washed with distilled waterand dried, then stained with uranylacetate and lead citrate and examined with Hitachi HU-12A and H-800 electron microscopes.

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