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.

scholarly journals Ultrastructural localization of nucleic acids through several cytochemical techniques on osmium-fixed tissues: comparative evaluation of the different labelings.

1984 ◽  
Vol 32 (11) ◽  
pp. 1185-1191 ◽  
Author(s):  
M Bendayan ◽  
E Puvion
Keyword(s):  
Nucleic Acids ◽  
Osmium Tetroxide ◽  
Phosphotungstic Acid ◽  
Sodium Tungstate ◽  
Ethylenediaminetetraacetic Acid ◽  
High Specificity ◽  
Ultrastructural Localization ◽  
Gold Complex ◽  
Sodium Metaperiodate ◽  
Tissue Sections

Several cytochemical techniques, such as sodium tungstate, acid hydrolysis phosphotungstic acid (HAPTA), ethylenediaminetetraacetic acid (EDTA), RNase-gold, and osmium-ammine, have been applied for the ultrastructural demonstration of nucleic acids on sections of tissues fixed in glutaraldehyde postfixed with osmium tetroxide and embedded in Epon. In order to obtain specific results, the sections had to be treated with sodium metaperiodate prior to performing the labeling protocol. The results for each method were identical to those obtained on nonosmicated tissues; the main difference being the enhancement in the ultrastructural preservation, which allowed for higher resolution. In addition to these techniques, and for comparative evaluations, DNA was also revealed by the DNase-gold approach on nonosmicated tissue sections. The consistency in the results, obtained over the nucleus with either EDTA or the RNase-gold complex for revealing RNA and those obtained with either osmium-ammine or DNase-gold for revealing DNA, supports the high specificity of the RNase-gold, DNase-gold, and osmium-ammine techniques. Furthermore, these results demonstrate the possibility of performing various cytochemical techniques on tissues processed for routine electron microscopy.

scholarly journals Rapid-freezing and malachite green-acrolein-osmium tetroxide freeze-substitution fixation improve visualization of extracellular lipids in rat incisor pre-dentin and dentin.

1987 ◽  
Vol 35 (4) ◽  
pp. 427-433 ◽  
Author(s):  
M Goldberg ◽  
F Escaig
Keyword(s):  
Malachite Green ◽  
Osmium Tetroxide ◽  
Freeze Substitution ◽  
Rapid Freezing ◽  
Dense Network ◽  
Rat Incisor ◽  
Unequal Distribution ◽  
Tissue Sections ◽  
Extracellular Matrix Components ◽  
Matrix Components

Rat incisor tissue sections were fixed by a modified version of the malachite green-aldehyde method (MGA) composed of rapid-freezing, malachite green-acrolein staining, and osmium tetroxide freeze-substitution (Fr.MGAO). In the pre-dentin, a thick, dense network of branched fibrous structures was observed. Cryotechniques allowed visualization of complexes about twice as thick and dense as the aggregates visualized on MGA-treated sections. Pretreatment of rapid-frozen samples with methanol before freeze-substitution fixation and staining prevented staining of the complexes otherwise revealed by the Fr.MGAO method. Electron-dense material stained by this procedure resisted de-mineralization with EDTA, while intramitochondrial granules and dentin crystallites were dissolved. EDTA treatment demonstrated unequal distribution of Fr.MGAO staining in dentin in the form of tiny dots underlining the collagen fibers. These results support the concept that rapid-freezing, followed by staining and freeze-substitution fixation, improves preservation of the phospholipids visualized as extracellular matrix components in pre-dentin and dentin of rat incisors.

THE SECRETORY PATHWAY IN PLATELETS STUDIED BY CRYO-FIXATION

1987 ◽  
Author(s):  
E morqenstern ◽  
H Patscheke
Keyword(s):  
Osmium Tetroxide ◽  
Secretory Pathway ◽  
Freeze Substitution ◽  
Human Platelets ◽  
Dense Matrix ◽  
Compound Exocytosis ◽  
Ultrathin Sections ◽  
Granule Secretion ◽  
Large Compound ◽  
Electron Dense Matrix

It is widely held, that the constituents packed in the a -granules are released by stimulated platelets via the surface connected system (SCS). By means of the fast-freezing and freeze substitution technique (which allow the investigation of membrane fusion) we found a secretory pathway in platelets (compound exocytosis) without an involvement of the SCS during the release of a-granules. To study the process of a-granule secretion human platelets concentrated in citrated blood plasm were stimulated with thrombin or collagen. 20 - 120 seconds after stimulation the platelets were rapidly frozen with a metal-mirror attachment to the KF 80 cryofixation unit (REICHERT-JUNG). Using plastic spacers droplets of the PRP were slammed against a copper block at 80 K at a rate of 0.2 m/sec. After cryofixation the specimens were transferred (in liquid nitrogen) into a Cs-auto cryosubstitution unit (REICHERT-JUNG). Cryosubstitution was programmed for 48h at 193 K in acetone with 4% osmium tetroxide. The temperature went automatically up to room temperature at a rate of 10 K/h. The specimens were embedded in araldite. The analysis of serial ultrathin sections of platelets in different phases of exocytosis revealed the following. a -granules in apposition showed different stages of swelling and dispersal of their electron dense matrix. Membrane appositions were also found between a -granules. The contraction of a sphere of microfilaments and microtubules during stimulation seemed to support this process. On the other hand this internal contraction prevented most of the a-granules from contacting with the plasmalemma. We observed fusion between swollen -granules in apposition and the plasmalemma and swollen and unswollen a -granules. Thus, large compound granules were formed frequently before fusion of the secretory organelles with the plasmalemma took place. These observations suggested that a -granules in stimulated platelets performed a compound exocytosis after swelling. The process seemed to start with the apposition of a -granule membranes to the plasmalemma. It cannot yet be answered whether the swelling of the granules is due to an osmotically driven influx of water or due to an influx after microfusion.Supported by DFG, Grant Mo 124/2-4

scholarly journals Methods and Principles of Fixation by Freeze-Substitution

1958 ◽  
Vol 4 (5) ◽  
pp. 593-602 ◽  
Author(s):  
Ned Feder ◽  
Richard L. Sidman
Keyword(s):  
Low Temperatures ◽  
Room Temperature ◽  
Osmium Tetroxide ◽  
Picric Acid ◽  
Freeze Substitution ◽  
Tissue Structure ◽  
Chemical Agent ◽  
Rapid Freezing ◽  
Chemical Mechanisms ◽  
Accurate Localization

Freeze-substitution is based on rapid freezing of tissues followed by solution ("substitution") of ice at temperatures well below O°C. A 1 to 3 mm. specimen was thrown into 3:1 propane-isopentane cooled by liquid nitrogen to -175°C. (with precautions). The frozen tissue was placed in substituting fluid at -70°C. for 1 week to dissolve ice slowly without distorting tissue structure. Excess substituting agent was washed out, and the specimen was embedded, sectioned, and stained conventionally. For best morphological and histochemical preservation, substituting fluids should in general contain both chemical fixing agent and solvent for ice, e.g., 1 per cent solutions of osmium tetroxide in acetone, mercuric chloride in ethanol, and picric acid in ethanol. Preservation of structure was poorer after substitution in solvent alone. Evidence was obtained that the chemical agent fixes tissue at low temperatures. The chemical mechanisms of fixation are probably similar to those operating at room temperature: new chemical cross-linkages, which contain the fixing agent, join tissue constituents together. This process is distinguished from denaturation by pure solvents. Freeze-substitution has many advantages, particularly the preservation of structure to the limit of resolution with the light microscope, and the accurate localization of many soluble and labile substances.

Uranyl Acetate as a Fixative—from pH 2.0 to 8.0

1971 ◽  
Vol 29 ◽  
pp. 490-491
Author(s):  
V. R. Mumaw ◽  
B. L. Munger
Keyword(s):  
Electron Microscopy ◽  
Oxalic Acid ◽  
Osmium Tetroxide ◽  
Uranyl Acetate ◽  
En Bloc ◽  
Tissue Sections ◽  
Normal Rat ◽  
Lead Hydroxide ◽  
Ultrathin Sections ◽  
Rat Tissue

Numerous applications utilizing uranyl acetate as an electron stain for electron microscopy have been described. Uranyl acetate has become a routine stain used in conjunction with lead hydroxide for staining ultrathin sections. En bloc staining with uranyl acetate following osmium tetroxide post-fixation produces undesirable effects on some cytoplasmic components, especially glycogen. Recent studies using uranyl acetate as a fixative and en bloc stain at pH 7.2 before osmification has shown uranyl acetate to have desirable fixation and staining qualities. Tissues treated with uranyl acetate at a pH of 2.0-8.0 were studied. Normal rat tissue was fixed in Karnovsky's paraformaldehyde-glutaraldehyde fixative. The tissue was post-fixed in 0.5% uranyl acetate in water at pH 2.0 and 0.5% uranyl acetate in 0.1M s-collidine with 0.01M oxalic acid at pH 4, pH 6.0, pH 7.2, and pH 8.0 for 1 hour at 4°C. Following several rinses of 0.1M s-collidine buffer, the tissues were treated with 1.33% osmium tetroxide 1 hour at 4°C followed by rapid dehydration in ethanol and embedded in Durcupan ACM. Tissue sections were stained with lead hydroxide.

Freeze substitution studies on cultured human fibroblasts

1989 ◽  
Vol 47 ◽  
pp. 1008-1009
Author(s):  
Julian P. Heath ◽  
Donna Turner
Keyword(s):  
Osmium Tetroxide ◽  
Single Cells ◽  
Human Fibroblasts ◽  
Three Dimensional ◽  
Freeze Substitution ◽  
Uranyl Acetate ◽  
Lung Fibroblasts ◽  
Rapid Freezing ◽  
Thin Sections ◽  
Cultured Human Fibroblasts

We are using rapid freezing and freeze substitution to study the three dimensional organisation of membrane systems and cytoskeletal filaments in motile fibroblasts. This study has two objectives: first, to provide material for structural and immunocytochemical analysis of membrane-cytoskeletal interactions in cells that have been preserved with minimum artefact (1,2,3) and second, to refine and develop existing rapid freezing and freeze substitution techniques to allow for the study of single cells that have been experimentally manipulated and observed by digital video microscopy before fixation.The cells used were human lung fibroblasts (IMR90) either growing on Lux Thermanox coverslips or as pelleted suspensions. The cells were slam frozen on a Med-Vac Cryo Press against a liquid nitrogen cooled copper block. Coverslips were trimmed to 2 x 2 mm in size, excess fluid was drained off, and they were placed on top of a 1mm thick gelatin cushion on an aluminium planchette. For cell suspensions, 3 ul was placed on top of the gelatin cushion. Frozen samples were placed in acetone containing 1% osmium tetroxide for 72 hours at 192 K, wanned to 253 K for 4 hours, and then brought to room temperature. The samples were rinsed in acetone and embedded in Spurr’s resin. Thin sections were cut on a RMC6000 ultramicrotome, stained in uranyl acetate and Reynolds' lead citrate and photographed on a Philips EM410 electron microscope at 60 keV.

scholarly journals Immunoelectron microscopic labeling of immunoglobulin in plasma cells after osmium fixation and epoxy embedding.

1985 ◽  
Vol 33 (12) ◽  
pp. 1212-1218 ◽  
Author(s):  
S A Hearn ◽  
M M Silver ◽  
J A Sholdice
Keyword(s):  
Epoxy Resin ◽  
Osmium Tetroxide ◽  
Secretory Pathway ◽  
Plasma Cells ◽  
Protein A ◽  
Ultrastructural Localization ◽  
Cell Organelles ◽  
Human Tonsil ◽  
Sodium Metaperiodate ◽  
Gold Label

Previous studies have found that immunoglobulin cannot be immunolabeled in tissues prepared for electron microscopy by usual methods. To test this conclusion, we used a protein A-gold postembedding immunolabeling method on tissues that were fixed in glutaraldehyde, post-fixed in osmium tetroxide, and embedded in epoxy resin; sections were pretreated with sodium metaperiodate. A variety of common fixation protocols were also used and the most suitable conditions for immunolabeling were determined. This technique permitted the ultrastructural localization of immunoglobulin light chains in optimally preserved and contrasted plasma cells from human tonsil, lymph nodes, plasmacytomas, and a renal biopsy. We were able to demonstrate multiple antigens in the same tissue and label antigens in tissues that had been stored for many years in epoxy resin. The technique allows quantitation of the gold label over plasma cell organelles and therefore gives information about the immunoglobulin secretory pathway in these cells. We found that the protein A-gold procedure compares favorably in technical ease with the immunoperoxidase, avidin-biotin peroxidase, and immunoglobulin-colloidal gold immunolabeling methods, and has added advantages in allowing precise localization and quantitation of the labeled antigen.

Structural Variations Along the Length of Multiply Innervated Muscle Fibers in Rat Inferior Rectus—A Serial Analysis

1981 ◽  
Vol 39 ◽  
pp. 500-501
Author(s):  
J. Davidowitz ◽  
D. J. Chiarandini ◽  
G. H. Philips ◽  
G. M. Breinin
Keyword(s):  
Osmium Tetroxide ◽  
Sampling Methods ◽  
Wistar Rat ◽  
Muscle Fiber Types ◽  
Fiber Types ◽  
Structural Variations ◽  
Inferior Rectus ◽  
Serial Sampling ◽  
Ultrathin Sections ◽  
Standard Sampling

Morphological definitions of muscle fiber types have generally been based on the tacit assumption that the structure of a given fiber is essentially constant along it length. Recently, however, the serial sampling of rabbit extraocular muscle (EOM) has shown that some multiply innervated fibers (MIFs) display systematic variation of both the sarcoplasmic reticulum (SR) and mitochondrial content. The purpose of the present study was to determine whether systematically varying MIFs occur in rat EOM as well: such fibers had not been detected in previous studies of rat EOM by means of standard sampling methods. A young (175 gm) white Wistar rat was perfused with 2% paraformaldehyde-2% glutaraldehyde in phosphate buffer. The inferior rectus was removed intact, fixed in 4% glutaraldehyde, postfixed in 1% osmium tetroxide, dehydrated in graded ethanols, and embedded whole in Epon 812. The muscle was serially sectioned at 15u, and 114 1u sections and 42 ultrathin sections were recut from the original thick sections for sequential samplings.

Development of spermatia in the red alga Caloglossa leprieurii preserved for TEM by freeze substitution

1990 ◽  
Vol 48 (3) ◽  
pp. 668-669
Author(s):  
Wilma L. Lingle ◽  
David Porter ◽  
Marshall Darley
Keyword(s):  
Red Algae ◽  
Room Temperature ◽  
Osmium Tetroxide ◽  
Freeze Substitution ◽  
Red Alga ◽  
Uranyl Acetate ◽  
Rapid Freezing ◽  
Liquid Propane ◽  
Laboratory Cultures ◽  
To Come

The red algae have been difficult to preserve for TEM observation. In an effort to overcome the limitations of conventional, aqueous fixations, we used rapid freezing techniques that are superior in preserving delicate and transient membrane features. Laboratory cultures of the red alga Caloglossa leprieurii were prepared for TEM by rapid freezing in liquid propane followed by substitution at -80° in 2% osmium tetroxide and 0.05% uranyl acetate in dry acetone for 65 hours. While in substitution fluid, the tissue was allowed to come to room temperature over a 6 hour period. The fluidwas replaced with dry acetone, followed by two 30 minute washes. Infiltration with Embed 812 without accelerator was performed at 4° on a tumbler in increments of 12.5% changed every 12 hours. Five percent increments were utilized from 75% to 100%. Three 24 hour exchanges with 100% resin including accelerator took place alternately under vacuum at room temperature for 8 hours, then at 4°on a tumbler for 16 hours. The tissue was cured at 60°.

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.

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