scholarly journals 2D and 3D immunogold localization on (epoxy) ultrathin sections with and without osmium tetroxide

2020 ◽  
Vol 83 (6) ◽  
pp. 691-705 ◽  
Author(s):  
Jennifer Flechsler ◽  
Thomas Heimerl ◽  
Carolin Pickl ◽  
Reinhard Rachel ◽  
York‐Dieter Stierhof ◽  
...  
Keyword(s):  
Osmium Tetroxide ◽  
Immunogold Localization ◽  
Ultrathin Sections ◽  
2D And 3D

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.

The Identification of Phospholipid Micelles in Glutaraldehyde-Urea Embedded Tissue

1975 ◽  
Vol 33 ◽  
pp. 494-495
Author(s):  
Daniel C. Pease
Keyword(s):  
Electron Micrographs ◽  
Lung Tissue ◽  
Osmium Tetroxide ◽  
Uranyl Acetate ◽  
Type Ii ◽  
Lead Citrate ◽  
Phospholipid Micelles ◽  
Type Ii Pneumocytes ◽  
Ultrathin Sections ◽  
Polar Water

It is reasonable to think that phospholipid micelles should be visible and identifiable in electron micrographs of ultrathin sections if only they can be preserved throughout the embedding process. The development of highly polar, water-containing, aminoplastic embedments has made this a likely possibility. With this in mind, an investigation of the lecithin-secreting, Type II pneumocytes of the lung is underway.Initially it has been easiest to recognize phospholipid micelles in lung tissue fixed first with glutaraldehyde, and then secondarily exposed to osmium tetroxide. However, the latter is not a necessary concomitant for micellar preservation. Conventional uranyl acetate and lead citrate staining is finally applied. Importantly, though, the micelles have been most easily seen in tissue embedded in 507. glutaraldehyde polymerized with urea, as described in detail by D.C. Pease and R.G. Peterson (J. Ultra- struct. Res., 41, 133, 1972). When oriented appropriately, the micellar units are seen as tiny, bilayer plates.

Portein-gold labelling of ultrathin sections of wheat spot mosaic-affected wheat plants

1990 ◽  
Vol 48 (3) ◽  
pp. 680-681
Author(s):  
K. S. Zaychuk ◽  
M. H. Chen ◽  
C. Hiruki
Keyword(s):  
Osmium Tetroxide ◽  
Rnase A ◽  
Leaf Ultrastructure ◽  
Double Membrane ◽  
Young Root ◽  
Leaf Tissues ◽  
Membrane Bound ◽  
Ultrathin Sections ◽  
Gold Labelling ◽  
Electron Dense Core

Wheat spot mosaic (WSpM), which frequently occurs with wheat streak mosaic virus was first reported in 1956 from Alberta. Singly isolated, WSpM causes chlorotic spots, chlorosis, stunting, and sometimes death of the wheat plants. The vector responsible for transmission is the eriophyid mite, Eriophyes tulipae Kiefer. The examination of leaf ultrastructure by electron microscopy has revealed double membrane bound bodies (DMBB’s) 0.1-0.2 μm in diameter. Dispersed fibrils within these bodies suggested the presence of nucleic acid. However, neither ribosomes characteristic of bacteria, mycoplasma and the psittacosis group of organisms nor an electron dense core characteristic of many viruses was commonly evident.In an attempt to determine if the DMBB’s contain nucleic acids, RNase A, DNase I, and lactoferrin protein were conjugated with 10 nm colloidal gold as previously described. Young root and leaf tissues from WSpM-affected wheat plants were fixed in glutaraldehyde, postfixed in osmium tetroxide,and embedded in Spurr’s resin.

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 A METHOD FOR INTRACELLULAR AUTORADIOGRAPHY IN THE ELECTRON MICROSCOPE

1961 ◽  
Vol 10 (4) ◽  
pp. 577-587 ◽  
Author(s):  
M. H. Silk ◽  
A. O. Hawtrey ◽  
I. M. Spence ◽  
J. H. S. Gear
Keyword(s):  
Electron Microscope ◽  
Osmium Tetroxide ◽  
Red Light ◽  
Kidney Cells ◽  
Sodium Thiosulphate ◽  
Aqueous Sodium ◽  
Fine Wire ◽  
Ultrathin Sections ◽  
Bromine Vapor ◽  
Silver Metal

A technic is described for high resolution intracellular autoradiography in the electron microscope. Cultures of LLC-MK2 monkey kidney cells were incubated for 72 hours in a medium containing 0.4 µcurie per ml of thymidine-H3. After labeling, the cells were fixed with osmium tetroxide and embedded in methacrylate. Ultrathin sections of the labeled tissue were taken up on Formvar-coated and carbon-stabilized electron microscope grids. A 150 to 450 A layer of silver metal was then evaporated onto the tissue. The coated grids were exposed to bromine vapor for 1.5 to 2 minutes under red light, allowed to dry for 1 minute, and then covered with a thin film of 1 per cent aqueous gelatin applied by means of a fine wire loop lowered over the grid supported on a glass peg. For autoradiographic exposure, the grids were stored 50 days in a light-proof container at 4°C with calcium chloride desiccant. Development was carried out for 5 minutes at 20°C in Promicrol (May and Baker, England) diluted 1:1 with water, followed by a 1 minute water wash and fixation for 2.5 minutes in 15 per cent aqueous sodium thiosulphate. After removal of the gelatin by immersion for 16 hours in water at 37°C, the autoradiograms were dried and examined in the electron microscope. Ultrastructural detail was fairly well defined and the cytoplasm of each labeled cell was covered with an electron opaque deposit of silver, suggesting that a polynucleotide containing thymidine may be synthesized in the cytoplasm. The matter is discussed.

scholarly journals ENZYMATIC DIGESTION IN THE CYTOCHEMICAL DEMONSTRATION OF GLYCOGEN

1967 ◽  
Vol 15 (1) ◽  
pp. 14-20 ◽  
Author(s):  
FORTUNATO ROSA ◽  
FRANK B. JOHNSON
Keyword(s):  
Gastric Mucosa ◽  
Epoxy Resin ◽  
Liver Tissue ◽  
Osmium Tetroxide ◽  
Periodic Acid ◽  
Enzymatic Digestion ◽  
Selective Removal ◽  
Cytochemical Demonstration ◽  
Ultrathin Sections

Mounted ultrathin sections of rat fetal gastric mucosa and liver tissue, fixed in osmium tetroxide or glutaraldehyde and embedded in epoxy resin, are suitable for the cytochemical demonstration of glycogen by staining either with lead or by the periodic acid-thiosemicarbazide procedure. Enzymatic digestion with saliva or α-amylase can be performed on mounted sections of similarly treated tissues for the selective removal of glycogen prior to staining.

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

Myocardial Alterations in Animals Following High Altitude Exposure

1968 ◽  
Vol 26 ◽  
pp. 114-115
Author(s):  
M. B. Bischoff ◽  
W. D. Dean ◽  
T. J. Bucci ◽  
L . A. Frics
Keyword(s):  
Pulmonary Artery ◽  
Right Ventricular ◽  
High Altitude ◽  
Sea Level ◽  
Osmium Tetroxide ◽  
Ultrastructural Level ◽  
Uranyl Acetate ◽  
Heart Weight ◽  
Morphological Evidence ◽  
Ultrathin Sections

There is a paucity of morphological evidence, particularly at the ultrastructural level, to correlate with tile physiological effects of chronic exposure to high altitude. In this study the myocardium of dogs and rabbits subjected to five months exposure at an altitude of 14,110 feet was compared to that of animals residing at sea level. The animals kept at 14,110 feet were shipped from sea level (160 feet) witnout acclimatization at intermediate altitudes.The physiological parameters including the hematologic changes, pulmonary artery pressures, pulmonary artery oxygen saturations, electrocardiograph changes, right ventricular weight to body weight and right ventricular weight to total heart weight ratios observed in these animals have been reported previously.The animals were necropsied at the end of the five months. The myocardium was quickly removed, cut into small pieces and immersed in 1% osmium tetroxide buffered with veronal acetate. Following fixation the tissues were dehydrated in ascending ethanol concentrations followed by propylene oxide and embedded in Epon 812. Ultrathin sections were doubly stained in uranyl acetate and lead citrate.

Enzyme Digestion of Epon Embedded Akinetic Cilia

1975 ◽  
Vol 33 ◽  
pp. 688-689
Author(s):  
R. A. Hess ◽  
R. J. Thurston ◽  
K. H. Kilburn
Keyword(s):  
Osmium Tetroxide ◽  
Tap Water ◽  
Freshwater Mussel ◽  
Enzyme Digestion ◽  
Ultrastructural Studies ◽  
Gill Filaments ◽  
Hyperosmolar Solutions ◽  
Ultrathin Sections

Terminal gill cilia of the freshwater mussel Unio stop beating when exposed to hyperosmolar solutions. Ultrastructural studies of the cilia during akinesia showed the absence of the 9+2 doublet microtubules and the ciliary matrix was amorphous and extremely electron dense (Fig. 2). It seemed possible that the axonemal microtubules might not be disrupted by shock-stopping cilia, but rather were obscured by the formation of the pleomorphic matrices. This hypothesis was tested using a modified enzymatic proceedure which has been shown to specifically digest spermatozoa flagellar microtubules in Epon ultrathin sections.Terminal gill filaments with control cilia beating in aged tap water and akinetic cilia which had been exposed to 0.154 M NaCl were fixed at 22°C for 30 min in 3% phosphate buffered (pH 7.35) glutaraldehyde and post-fixed at 22°C for 30 min in 2% buffered osmium tetroxide. The gills were rinsed in buffer, dehydrated in ethanol and embedded in Epon 812.

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