A simple technique for staining of cell membranes with imidazole and osmium tetroxide.

1995 ◽  
Vol 43 (10) ◽  
pp. 1079-1084 ◽  
Author(s):  
G Thiéry ◽  
J Bernier ◽  
M Bergeron
Keyword(s):  
Osmium Tetroxide ◽  
Cell Membranes ◽  
Cell Organelles ◽  
Unsaturated Lipids ◽  
Transmission Electron ◽  
Glutaraldehyde Solution ◽  
Kidney Liver ◽  
The Relationship ◽  
Resin Penetration

We describe a simple new technique based on the affinity of imidazole and osmium tetroxide for unsaturated lipids. Organs (e.g., kidney, liver, intestine) were perfused in vivo with a glutaraldehyde solution. Tissue fragments were then immersed in a solution containing imidazole and OsO4 and are further stained with a double lead and copper citrate solution. Ultra-thin (0.06 microns) or thick (0.1-0.3 microns) sections were observed with transmission electron microscopy (80-100 kV). The method presented permits excellent visualization of cell membranes (e.g., endoplasmic reticulum, endocytotic apparatus) because it favors good resin penetration and the alkaline pH preserves cell volume. A better stereomicroscopic analysis of the relationship between cell organelles can be carried out with thick sections. The imidazole/osmium can be used routinely because the technical steps are easy and simple to follow. Furthermore, it can complement other cytochemical 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.

Variations in Sample Preparation that Affect Contrast Enhancement by Lead Aspartate

1980 ◽  
Vol 38 ◽  
pp. 654-655
Author(s):  
J.M. Yoshiyama ◽  
D. Goff ◽  
J. Walton
Keyword(s):  
Electron Microscopy ◽  
Contrast Enhancement ◽  
Osmium Tetroxide ◽  
Great Success ◽  
En Bloc ◽  
Transmission Electron ◽  
Wash Buffer ◽  
Glutaraldehyde Solution ◽  
Uniform Diameter ◽  
Made In

We recently described a new en bloc lead contrast stain for transmission electron microscopy (Walton, 1979). Several laboratories report great success with lead aspartate staining but some others have found that its contrast enhancement is insufficient. We undertook the present study to determine how differences in sample protocol affect lead aspartate contrast enhancement, making a strict effort to obtain comparable samples. Gastrointestinal duodenum provided a tissue of uniform diameter. A 1" length of duodenum was flushed with warm saline and fixed in 4% paraformaldehyde-1% glutaraldehyde solution buffered at pH 7.3 with 0.1 M cacodylate. After a 2 hr fixation period, the sample was cross-sectioned into blocks 1.0 mm thick. Samples were then post-fixed with 1% osmium tetroxide buffered with veronal acetate at pH 7.3, washed, stained with lead aspartate, rinsed, dehydrated, and embedded. Standard staining conditions were pH 5.5 lead aspartate heated to 60°C for 1 hr. This procedure was varied by adjusting either the pH of lead aspartate, the staining time, or the temperature. Additional variations were made in the dehydration times, osmium fixation times, and choice of wash buffer. Silver sections were cut from all samples. Micrographs were taken using uniform settings at either high or low magnifications. All negatives were developed and printed under the same conditions.

Ultrastructural Study of the Cecal Epithelium in Normal Guinea Pig

1977 ◽  
Vol 35 ◽  
pp. 650-651
Author(s):  
Juan Mora-Galindo ◽  
Adolfo Martínez-Palomo
Keyword(s):  
Guinea Pig ◽  
Osmium Tetroxide ◽  
Animal Species ◽  
Freeze Fracture ◽  
Microbial Diseases ◽  
Transmission Electron ◽  
Glutaraldehyde Solution ◽  
Ultrastructural Characteristics ◽  
Means Of Transmission ◽  
Cecal Mucosa

The use of guinea pig cecum in experimental amebiasis and other parasitic and microbial diseases, makes imperative to have a clear knowledge of its normal ultrastructure in order to evaluate properly the effects that the microorganisms may produce. Although several reports have appeared in the literature concerning the normal ultrastructure of the small and large intestines in various animal species including the human, it is difficult to extrapolate those descriptions to all animal species. The aim of the present work was to describe the ultrastructural characteristics of the normal guinea pig cecal mucosa, by means of transmission electron microscopy (TEM), scanning electron micro scopy (SEM), and freeze-fracture technics. Representative fragments of cecum from normal anesthetized guinea pigs were obtained through laparotomy and immediately fixed in 1% buffered osmium tetroxide and processed for TEM. Some samples were fixed in a 2.5% buffered glutaraldehyde solution and processed for SEM and freeze-fracture.

Cell organelles at uncoated cryofractured surfaces as viewed with the scanning electron microscope

1976 ◽  
Vol 21 (1) ◽  
pp. 47-58
Author(s):  
P.S. Woods ◽  
M.C. Ledbetter
Keyword(s):  
Osmium Tetroxide ◽  
Cell Organelles ◽  
Thin Sections ◽  
3 Dimensional ◽  
Transmission Electron ◽  
Uncoated Surface ◽  
Surface Irregularities ◽  
Coating Methods ◽  
Silver Paint ◽  
Scanning Electron

A method of direct visualization of cell organelles by scanning electron microscopy (SEM) is described. Plant and animal tissues fixed in glutaraldehyde and osmium tetroxide are treated with the ligand thiocarbohydrazide and a second osmium tetroxide solution, to increase their osmium content. Tissues are then dehydrated, infiltrated with an epoxy monomer, and together solidified with dry ice and fractured. The pieces are transferred to pure acetone, critical-point dried, attached to stubs with silver paint and viewed by SEM. The ligating procedure increases the osmium concentration at its original bonding site sufficiently to render the tissue electrically conductive, thus obviating the need for metallic coating. he organelles at the fractured surface are revaled in relation to their osmium incorporation rather than by surface irregularities as with coating methods. The image derived from the uncoated surface approaches in resolution that of transmission electron micrographs of thin sections. A protion of the image arising from a small distance below the surface, while at progressively lower resolution, provides some 3-dimensional information about cell fine structure.

Ultrastructural Correlations between Clonal Human Tumor Colonies and in Vivo Neoplasms

1982 ◽  
Vol 40 ◽  
pp. 210-211
Author(s):  
M.J. Murphy ◽  
R.R. Price ◽  
J.C. Sloman
Keyword(s):  
Cultured Cells ◽  
Human Tumor ◽  
Cell Type ◽  
Tumor Mass ◽  
Initial Cell ◽  
Cloning Assay ◽  
Human Tumor Cloning ◽  
The Relationship

The in vitro human tumor cloning assay originally described by Salmon and Hamburger has been applied recently to the investigation of differential anti-tumor drug sensitivities over a broad range of human neoplasms. A major problem in the acceptance of this technique has been the question of the relationship between the cultured cells and the original patient tumor, i.e., whether the colonies that develop derive from the neoplasm or from some other cell type within the initial cell population. A study of the ultrastructural morphology of the cultured cells vs. patient tumor has therefore been undertaken to resolve this question. Direct correlation was assured by division of a common tumor mass at surgical resection, one biopsy being fixed for TEM studies, the second being rapidly transported to the laboratory for culture.

Destruction of Actin Filaments by Osmium Tetroxide

1977 ◽  
Vol 35 ◽  
pp. 466-467
Author(s):  
P. Maupin-Szamier ◽  
T. D. Pollard
Keyword(s):  
Actin Filaments ◽  
Time Course ◽  
Osmium Tetroxide ◽  
Rabbit Muscle ◽  
Muscle Actin ◽  
Sodium Phosphate Buffer ◽  
Transmission Electron ◽  
The Difference ◽  
Rates Of Decay ◽  
Short Time

We have studied the destruction of rabbit muscle actin filaments by osmium tetroxide (OSO4) to develop methods which will preserve the structure of actin filaments during preparation for transmission electron microscopy.Negatively stained F-actin, which appears as smooth, gently curved filaments in control samples (Fig. 1a), acquire an angular, distorted profile and break into progressively shorter pieces after exposure to OSO4 (Fig. 1b,c). We followed the time course of the reaction with viscometry since it is a simple, quantitative method to assess filament integrity. The difference in rates of decay in viscosity of polymerized actin solutions after the addition of four concentrations of OSO4 is illustrated in Fig. 2. Viscometry indicated that the rate of actin filament destruction is also dependent upon temperature, buffer type, buffer concentration, and pH, and requires the continued presence of OSO4. The conditions most favorable to filament preservation are fixation in a low concentration of OSO4 for a short time at 0°C in 100mM sodium phosphate buffer, pH 6.0.

Scanning and Transmission Microscopy of Nematocyst Batteries in Epitheliomuscular Cells of Hydra

1971 ◽  
Vol 29 ◽  
pp. 410-411 ◽  
Author(s):  
Jane A. Westfall ◽  
S. Yamataka ◽  
Paul D. Enos
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Osmium Tetroxide ◽  
External Surface ◽  
Three Dimensional ◽  
Surface Structures ◽  
Transmission Microscopy ◽  
Transmission Electron ◽  
Freeze Dried ◽  
Scanning Electron

Scanning electron microscopy (SEM) provides three dimensional details of external surface structures and supplements ultrastructural information provided by transmission electron microscopy (TEM). Animals composed of watery jellylike tissues such as hydras and other coelenterates have not been considered suitable for SEM studies because of the difficulty in preserving such organisms in a normal state. This study demonstrates 1) the successful use of SEM on such tissue, and 2) the unique arrangement of batteries of nematocysts within large epitheliomuscular cells on tentacles of Hydra littoralis.Whole specimens of Hydra were prepared for SEM (Figs. 1 and 2) by the fix, freeze-dry, coat technique of Small and Màrszalek. The specimens were fixed in osmium tetroxide and mercuric chloride, freeze-dried in vacuo on a prechilled 1 Kg brass block, and coated with gold-palladium. Tissues for TEM (Figs. 3 and 4) were fixed in glutaraldehyde followed by osmium tetroxide. Scanning micrographs were taken on a Cambridge Stereoscan Mark II A microscope at 10 KV and transmission micrographs were taken on an RCA EMU 3G microscope (Fig. 3) or on a Hitachi HU 11B microscope (Fig. 4).

Cell Reactivity Pattern of the Guinea Pig Cecal Mucosa Evidenced by the ZIO Technique

1982 ◽  
Vol 40 ◽  
pp. 50-51
Author(s):  
Juan Mora-Galindo ◽  
Jorge Arauz-Contreras
Keyword(s):  
Guinea Pig ◽  
Phase Contrast ◽  
Osmium Tetroxide ◽  
Cell Types ◽  
Cell Reactivity ◽  
Transmission Electron ◽  
Neural Tissues ◽  
Maturation Stages ◽  
Zinc Iodide ◽  
Cecal Mucosa

The zinc iodide-osmium tetroxide (ZIO) technique is presently employed to study both, neural and non neural tissues. Precipitates depends on cell types and possibly cell metabol ism as well.Guinea pig cecal mucosa, already known to be composed of epithelium with cells at different maturation stages and lamina propria which i s formed by morphologically and functionally heterogeneous cell population, was studied to determine the pat tern of ZIO impregnation. For this, adult Guinea pg cecal mucosa was fixed with buffered 1.2 5% g 1 utara 1 dehyde before incubation with ZIO for 16 hours, a t 4°C in the dark. Further steps involved a quick sample dehydration in graded ethanols, embedding in Epon 812 and sectioning to observe the unstained material under a phase contrast light microscope (LM) and a transmission electron microscope (TEM).

Ultrastructural comparison of prolactin adenomas of pituitary in men and women

1988 ◽  
Vol 46 ◽  
pp. 346-347
Author(s):  
R.C. Caughey ◽  
U.P. Kalyan-Raman
Keyword(s):  
Endoplasmic Reticulum ◽  
Electron Microscope ◽  
Phosphate Buffer ◽  
Pituitary Adenomas ◽  
Osmium Tetroxide ◽  
Secretory Granules ◽  
Uranyl Acetate ◽  
En Bloc ◽  
Men And Women ◽  
Transmission Electron

Prolactin producing pituitary adenomas are ultrastructurally characterized by secretory granules varying in size (150-300nm), abundance of endoplasmic reticulum, and misplaced exocytosis. They are also subclassified as sparsely or densely granulated according to the amount of granules present. The hormone levels in men and women vary, being higher in men; so also the symptoms vary between both sexes. In order to understand this variation, we studied 21 prolactin producing pituitary adenomas by transmission electron microscope. This was out of a total of 80 pituitary adenomas. There were 6 men and 15 women in this group of 21 prolactinomas.All of the pituitary adenomas were fixed in 2.5% glutaraldehyde, rinsed in Millonig's phosphate buffer, and post fixed with 1% osmium tetroxide. They were then en bloc stained with 0.5% uranyl acetate, rinsed with Walpole's non-phosphate buffer, dehydrated with graded series of ethanols and embedded with Epon 812 epoxy resin.

Fine Structure of Planarian Neuroglial Cell

1976 ◽  
Vol 34 ◽  
pp. 188-189 ◽  
Author(s):  
Michio Morita ◽  
Jay Boyd Best
Keyword(s):  
Endoplasmic Reticulum ◽  
Ventral Nerve Cord ◽  
Osmium Tetroxide ◽  
Golgi Body ◽  
Nerve Fibers ◽  
Neuroglial Cell ◽  
Glutaraldehyde Solution ◽  
Deep Indentation ◽  
Cytoplasmic Processes ◽  
Longitudinal Nerve

The species of the planarian Dugesia dorotocephala was used as the experimental animal to study a neuroglial cell in the ventral nerve cord. Animals were fixed with 3% buffered glutaraldehyde solution and postfixed with 1% buffered osmium tetroxide.The neuroglial cell is multipolar, expanding into three or four cytoplasmic processes with many daughter branches. Some neuroglial processes are found to extend perpendicular to the longitudinal nerve fibers, whereas others are seen to be parallel to them. The nucleus of the neuroglial cell is irregular in shape and frequently has a deep indentation. Convex portions of the nucleus seem to be related to the areas from which cytoplasmic processes are extended. Granular endoplasmic reticulum (Fig. 4), Golgi body (Fig. 2), mitochondria (Figs. 1 and 2), microtubules (Fig. 4), and many glycogen granules are observable in the electron dense neuroglial cytoplasm. Neuroglial cells are also observed to contain various sizes of phagosomes and lipids (Fig. 2).

Sign in / Sign up

Export Citation Format

Share Document