Use of osmium tetroxide-potassium ferricyanide in reconstructing cells from serial ultrathin sections

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.

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The Identification of Phospholipid Micelles in Glutaraldehyde-Urea Embedded Tissue

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100116097 ◽

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.

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

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100160959 ◽

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.

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

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100167019 ◽

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.

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

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100161989 ◽

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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A METHOD FOR INTRACELLULAR AUTORADIOGRAPHY IN THE ELECTRON MICROSCOPE

The Journal of Cell Biology ◽

10.1083/jcb.10.4.577 ◽

1961 ◽

Vol 10 (4) ◽

pp. 577-587 ◽

Cited By ~ 26

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.

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ENZYMATIC DIGESTION IN THE CYTOCHEMICAL DEMONSTRATION OF GLYCOGEN

Journal of Histochemistry & Cytochemistry ◽

10.1177/15.1.14 ◽

1967 ◽

Vol 15 (1) ◽

pp. 14-20 ◽

Cited By ~ 22

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.

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