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.

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 A STUDY OF EXTRACELLULAR SPACE IN CENTRAL NERVOUS TISSUE BY FREEZE-SUBSTITUTION

1965 ◽  
Vol 25 (1) ◽  
pp. 117-137 ◽  
Author(s):  
A. Van Harreveld ◽  
Jane Crowell ◽  
S. K. Malhotra
Keyword(s):  
Tight Junctions ◽  
Extracellular Space ◽  
Nervous Tissue ◽  
Water Distribution ◽  
Circulatory Arrest ◽  
Freeze Substitution ◽  
Synaptic Cleft ◽  
Uranyl Acetate ◽  
Central Nervous Tissue ◽  
Silver Mirror

It was attempted to preserve the water distribution in central nervous tissue by rapid freezing followed by substitution fixation at low temperature. The vermis of the cerebellum of white mice was frozen by bringing it into contact with a polished silver mirror maintained at a temperature of about -207°C. The tissue was subjected to substitution fixation in acetone containing 2 per cent OsO4 at -85°C for 2 days, and then prepared for electron microscopy by embedding in Maraglas, sectioning, and staining with lead citrate or uranyl acetate and lead. Cerebellum frozen within 30 seconds of circulatory arrest was compared with cerebellum frozen after 8 minutes' asphyxiation. From impedance measurements under these conditions, it could be expected that in the former tissue the electrolyte and water distribution is similar to that in the normal, oxygenated cerebellum, whereas in the asphyxiated tissue a transport of water and electrolytes into the intracellular compartment has taken place. Electron micrographs of tissue frozen shortly after circulatory arrest revealed the presence of an appreciable extracellular space between the axons of granular layer cells. Between glia, dendrites, and presynaptic endings the usual narrow clefts and even tight junctions were found. Also the synaptic cleft was of the usual width (250 to 300 A). In asphyxiated tissue, the extracellular space between the axons is either completely obliterated (tight junctions) or reduced to narrow clefts between apposing cell surfaces.

scholarly journals Demonstration of Extracellular Space by Freeze-Drying in the Cerebellar Molecular Layer

1966 ◽  
Vol 1 (2) ◽  
pp. 223-228
Author(s):  
A. VAN HARREVELD ◽  
S. K. MALHOTRA
Keyword(s):  
Tight Junctions ◽  
Electron Micrographs ◽  
Extracellular Space ◽  
Freeze Drying ◽  
Circulatory Arrest ◽  
Molecular Layer ◽  
Freeze Substitution ◽  
Appreciable Amount ◽  
Nerve Fibres ◽  
Cerebellar Molecular Layer

In electron micrographs of the molecular layer of the mouse cerebellum frozen within 30 sec of circulatory arrest and subsequently dried at -79 °C an appreciable extracellular space was found between the axons of the granular cells. Tight junctions were regularly observed between pre- and postsynaptic structures and the enveloping glia cells. In micrographs of cerebellum frozen 8 min after decapitation the space between the axons was absent and tight junctions between the nerve fibres were almost exclusively encountered. The extracellular space of asphyxiated and non-asphyxiated tissue in electron micrographs of frozen-dried material is similar to the space in comparable tissues treated by freeze-substitution. These observations suggest that there is an appreciable amount of extracellular material in oxygenated, living tissue whichis taken up by cellular elements during asphyxiation.

scholarly journals VARIATIONS IN THE LOCATION AND SIZE OF PYROANTIMONATE PRECIPITATES IN THE IMMATURE RAT CEREBRAL CORTEX

1971 ◽  
Vol 19 (10) ◽  
pp. 591-604 ◽  
Author(s):  
S. M. SUMI
Keyword(s):  
Cerebral Cortex ◽  
Osmium Tetroxide ◽  
Rat Cerebral Cortex ◽  
Newborn Rat ◽  
Immature Rat ◽  
Marked Variability ◽  
Perfusion Fixation ◽  
Developing Rat ◽  
Potassium Pyroantimonate

The extracellular space in the developing rat cerebral cortex is quite large but varies considerably in extent depending on age, method of fixation and osmolarity of the fixative. Since Na+ is known to be extracellular in location, the potassium pyroantimonate technique of Komnick was utilized in the newborn rat in order to follow the movement of Na+ and water. However, the location and size of the pyroantimonate precipitates appeared to vary with the type of fixative solution and method of fixation. Thus, they were large and mainly intracellular with phosphate- or s-collidine-buffered glutaraldehyde after perfusion fixation, and mostly extracellular after immersion fixation. Following primary osmium tetroxide fixation, these particles were small and numerous with the aqueous solution, being diffusely present throughout the section, and large and fewer with the phosphate-buffered solution. Whether Na+ alone was being demonstrated could not be determined. Because of this marked variability it is concluded that definite statements as to the actual in vivo location of the Na+ in this preparation cannot be made.

Ultrastructural localization of neuropeptides in the rat brain

1978 ◽  
Vol 36 (2) ◽  
pp. 612-613
Author(s):  
F.W. Van Leeuwen
Keyword(s):  
Freeze Substitution ◽  
Ultrastructural Localization ◽  
Serial Sections ◽  
Microscopical Level ◽  
Electron Microscopical Level ◽  
Enzyme Method ◽  
Perfusion Fixation ◽  
Electron Microscopical ◽  
Unlabeled Antibody ◽  
Peroxidase Conjugate

In order to obtain specific and optimal ultrastructural localization of vasopressin and oxytocin in the hypothalamo-neurohypophyseal system of the rat, 2 staining procedures and several tissue treatments were evaluated using neurohypophyseal tissue. It appeared from these studies that post-embedding staining with the unlabeled antibody enzyme method developed by Sternberger allows greater dilution of the first antibody (anti-vasopressin, 1:4800) than the indirect procedure (1:320) using a peroxidase conjugate as second antibody. Immersion fixation with 4% formalin during 24 hours gave better results (general ultrastructure, immunoreactivity) than those obtained by perfusion fixation with 2.5% glutaraldehyde-1% paraformaldehyde or freeze substitution.Since no reliable specificity tests were performed at the electron microscopical level, tests were developed for antibodies against both vasopressin and oxytocin. For anti-vasopressin plasma neural lobes of homozygous Brattleboro rats, that are lacking vasopressin by a genet- ical defect, were used. For antibodies against both hormones serial sections were used that were alternately incubated with the antibodies.

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.

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.

Perfusion fixation of lungs for structure-function analysis: credits and limitations

1982 ◽  
Vol 53 (2) ◽  
pp. 528-533 ◽  
Author(s):  
H. Bachofen ◽  
A. Ammann ◽  
D. Wangensteen ◽  
E. R. Weibel
Keyword(s):  
Osmium Tetroxide ◽  
Function Analysis ◽  
Transpulmonary Pressure ◽  
Uranyl Acetate ◽  
Vascular Perfusion ◽  
Lung Weight ◽  
Physiological Variables ◽  
Isotonic Buffer ◽  
Perfusion Fixation

The quality of tissue preservation in lungs fixed by vascular perfusion has been reevaluated. Excised rabbit lungs inflated to 60% of total lung capacity were perfused (zone III conditions) with different but widely used fixatives. The effects of the perfusates on pertinent physiological variables have been assessed by a continuous monitoring, the effects on the pulmonary microstructure by qualitative and morphometric analysis of electron micrographs. Important results include the following. 1) Perfusions with isotonic glutaraldehyde at flow rates within the physiological range produce large increases of perfusion pressure and lung weight that reflect intracellular, interstitial, and intra-alveolar edema. 2) No edema occurs if glutaraldehyde is added to isotonic buffer solutions (total osmolarity 510 mosM). 3) Glutaraldehyde as sole perfusate does not fully eliminate the retractive force of lung tissue. Upon release of transpulmonary pressure the lungs retract by an indeterminable amount. 4) Satisfactory results can be obtained by sequential perfusion with osmium tetroxide and uranyl acetate or glutaraldehyde (510 mosM) followed by osmium tetroxide and uranyl acetate. The latter combination yields optimal preparations to study the alveolar and capillary architecture but causes a hyperosmotic volume loss of lung cells (cell shrinkage).

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