Uranyl Acetate En Bloc Staining to Complement Diagnostic Electron Microscopic Evaluation of Muscle Biopsies

1985 ◽  
Vol 43 ◽  
pp. 462-463
Author(s):  
Caughey R.C. ◽  
Kalyan-Raman U.P.
Keyword(s):  
Plasma Membranes ◽  
Uranyl Acetate ◽  
Staining Procedure ◽  
Lead Citrate ◽  
Ethanol Dehydration ◽  
Electron Microscopic ◽  
En Bloc ◽  
Microscopic Evaluation ◽  
Acetate Lead ◽  
Muscle Biopsies

Uranyl acetate en bloc staining is known to improve overall contrast and membrane preservation and removes glycogen from tissues. This staining procedure complements diagnostic electron microscopic evaluation by enhancing ultrastructural details. The ultrastructural structures enhanced are basement and plasma membranes, mitochondrial membrane and cristae, sarcoplasmic reticulum and T-tubule system. We wanted to study the usefulness of this technique over the conventional method in the study of muscle biopsy. Ultrastructurally, thirty muscle biopsies submitted to our Electron Microscopic Lab were routinely fixed in 2.5% glutaraldehyde and 1% 0S04, dehydrated in a graded series of ethanols, and embedded in Epon 812. Half of the blocks were processed and evaluated with post uranyl acetate/lead citrate staining. The other half were processed and evaluated by en bloc 0. 5% uranyl acetate/Walpole's buffer after 0S04 and before ethanol dehydration. The en bloc half were also stained with post uranyl acetate/lead citrate.

Nonspecific structures seen in diagnostic muscle biopsies

1987 ◽  
Vol 45 ◽  
pp. 846-847
Author(s):  
R. C. Caughey ◽  
U. P. Kalyan-Raman
Keyword(s):  
Electron Microscope ◽  
Transmission Electron Microscope ◽  
Osmium Tetroxide ◽  
Clinical History ◽  
Uranyl Acetate ◽  
Ethanol Dehydration ◽  
Tubular Aggregates ◽  
En Bloc ◽  
Transmission Electron ◽  
Muscle Biopsies

In a period of two years we have analyzed 50 muscle biopsies using the transmission electron microscope. Six nonspecific structures consisting of filamentous bodies, tubular aggregates, paracrystalline mitochondrial inclusions, honeycomb arrays, concentric laminated bodies, and finger print profiles were observed in 47 of 50 cases. In order to know the significance of these structures in muscle biopsies, we correlated their occurrence with their clinical history, histological findings, and histochemistry.The biopsies were initially fixed in 2.5% glutaraldehyde (pH. 7.5, 500 mOsm), then randomly minced and post fixed in 1% osmium tetroxide. All biopsies were processed with and without uranyl acetate en bloc staining in Walpole's buffer before ethanol dehydration. They were embedded in Epon 812 epoxy resin, sectioned, and stained with uranyl acetate and lead citrate before evaluation with a JEOL, JEM 100 C Transmission Electron Microscope. All grid squares of six different blocks were scanned to evaluate the ultra-structural pathology.

A double staining technique for improved contrast of thin sections from Spurr-embedded tissue

1978 ◽  
Vol 56 (1) ◽  
pp. 129-132 ◽  
Author(s):  
D. F. Bray ◽  
E. B. Wagenaar
Keyword(s):  
Potassium Permanganate ◽  
Staining Technique ◽  
Uranyl Acetate ◽  
Staining Procedure ◽  
Lead Citrate ◽  
Double Staining ◽  
Thin Sections ◽  
Citrate Method ◽  
Acetate Lead

A double staining procedure using potassium permanganate and lead citrate is described. The technique, involving treatment of sections with a 1% solution of potassium permanganate followed by lead citrate staining, is shown to be more effective in producing contrast in Spurr-embedded tissue than the conventional uranyl acetate – lead citrate method.

Electron-Microscopic localization of biotinylated gastrin bound to eosinophils in the rat stomach

1994 ◽  
Vol 52 ◽  
pp. 300-301
Author(s):  
Caroline A. Miller ◽  
David H. Nichols ◽  
Richard F. Murphy
Keyword(s):  
Electron Microscope ◽  
Phosphate Buffer ◽  
Uranyl Acetate ◽  
List Type ◽  
Cerium Chloride ◽  
Rat Stomach ◽  
Electron Microscopic ◽  
Human Sequence ◽  
Acetate Lead ◽  
Microscope Slides

Gastrin is a small peptide capable of both stimulating gastric acid secretion and acting as an enteric growth factor. Known functions of eosinophils in the rat stomach are related to immunological defense. Here we demonstrate the binding of biotinylated gastrin to rat stomach eosinophils in the electron microscope. Small pieces of stomach were fixed by immersion in 4% paraformaldehyde/0.1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4 for 1 hour. The tissue was then cryoprotected in 30% sucrose/0.1 M phosphate buffer, transferred to Tissue Tek OCT compound and frozen in isopentane cooled with liquid nitrogen. Transverse cryostat sections were cut at 25 μm, thawed in PBS and free floating sections exposed to 10−5 M biotinylated 1-17 gastrin (human sequence; Peninsula Labs) for 1 hour. Controls omitted the biotinylated gastrin from this step. Sections were then rinsed 3X in PBS and exposed to either:1).a 1:50 dilution of 10 nm Extravidin colloidal gold (Sigma) for 2 hours, or2).an avidin-biotin-alkaline phosphatase complex (ABC-AP;Vector) for 1 hour. A substrate solution containing cerium chloride was used to generate an electron dense reaction product.Sections from both procedures were postfixed in 1% OsO4 in 0.1 M phosphate buffer, rinsed and dehydrated. These were then flat embedded in EMbed 812 between two microscope slides coated with Liquid Release (both from Electron Microscopy Sciences).Polymerized sections were adhered to resin blocks using super glue, cut at 70-90 nm, stained with uranyl acetate/lead citrate and observed in a Philips CM-10 electron microscope.

Microscopic investigations of novel intracellular bodies of a Nocardia SP

1994 ◽  
Vol 52 ◽  
pp. 356-357
Author(s):  
J. L. Stites
Keyword(s):  
Uranyl Acetate ◽  
Electron Microscopic ◽  
En Bloc ◽  
Ribonucleic Acids ◽  
Transmission Electron ◽  
Nocardia Sp ◽  
Internal Constitution ◽  
Membrane Bound ◽  
Molecular Components ◽  
Protein Rich

A Nocardia sp.was found during an initial transmission electron microscopic (TEM) examination to have unusual intracellular bodies (ICB's) which do not appear to have been described previously in the literature. Most intracellular structures within bacteria have been classified as storage granules, a product of membrane invagination (i.e. mesosomes), or vacuoles. In bacteria there are no known intracellular membrane-bound organelles, and all internal membranes are invaginations of the unit membrane. Several microscopic-level examinations of the Nocardia sp. ICB's were initiated in order to determine their overall structure, classification, and internal constitution.Different TEM staining procedures were performed to determine possible molecular components of the ICB. In all of the staining protocols the ICB's showed a lack of electron density similar to the cell wall. Because the ICB's showed no affinity to any stain, it appeared they do not have strong positive charge (phosphotungstic acid), are not protein rich (en bloc uranyl acetate), lack glycogen and are not phosphate or sulphur rich (lead citrate), nor do they contain lipids or ribonucleic acids (osmium tetroxide).

Recovery of the Venous Wall from Leukocyte Induced Injury

1975 ◽  
Author(s):  
G. J. Stewart ◽  
W. G. M. Ritchie ◽  
P. R. Lynch
Keyword(s):  
Smooth Muscle ◽  
Surface Membrane ◽  
Uranyl Acetate ◽  
Surgical Trauma ◽  
Luminal Surface ◽  
Lead Citrate ◽  
Jugular Veins ◽  
Acetate Lead ◽  
Partial Covering ◽  
Venous Wall

Surgical trauma to tissue adjacent to canine jugular veins combined with brief local stasis caused massive leukocyte invasion of venous walls. This resulted in the entrapment of pockets of leukocytes between the endothelium and basement membrane and subsequent detachment of patches of endothelium.By an hour smooth muscle showed a decrease in myofibrils and an increase in rough endoplasmic reticulum and collagen lost its ability to stain with uranyl acetate-lead citrate.By 24 hours smooth muscle cells had undergone various changes and formed a partial covering layer over the surface of denuded areas, often by extending long “arms” from a bulky, fibroblast-like “body”. Various stages of de-differentiation and re-differentiation were abundant. By 72 hours these cells had become thinner and resembled the “immature” endothelium of Florey and others. However, the sheet was still discontinuous. By 7 days and thereafter to 28 days the endothelium was continuous and typical of “immature” endothelium morphologically but the surface membrane stained intensely in some instances.At 24 hours there was a perivascular zone of edema with cell ghosts and amorphous debris which decreased thereafter. Collagen staining was again typical by 7 days.These observations indicate that the luminal surface of blood veins can be initially repaired by the rapid de-differentiation and re-differentiation of smooth muscle rather than waiting for the ingrowth of endothelial cells from the margins of the denuded areas.(Supported by N. I. H. Grants ≠ HL 14217 and HL 08886.)

The fine structure of ascus development in Lasiobolus monascus (Pezizales)

1978 ◽  
Vol 56 (7) ◽  
pp. 862-872 ◽  
Author(s):  
James W. Kimbrough ◽  
Gerald L. Benny
Keyword(s):  
Fine Structure ◽  
Uranyl Acetate ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Stalk Cell ◽  
Lead Citrate ◽  
Electron Microscopic ◽  
Ultrathin Sections ◽  
Microscopic Inspection ◽  
Physical And Chemical

Ultrastructural and cytochemical studies on the ascus of Lasiobolus monascus are presented. Apothecia in various stages of development were obtained in culture and prepared for both light and electron microscopic observations. Ultrathin sections for electron microscopic inspection were often treated with silver methenamine to enhance wall characteristics. Ascus development was followed from fertilization to maturity.In this species, the ascogonium enlarges after fertilization to become the ascus mother cell. Two pores are present in the young ascus, one connecting it to the antheridium and another between the ascus and stalk cell. The ultrastructural features of these pores in the young and maturing ascus are described. During ascus enlargement, as many as four wall layers are found when poststained with silver methenamine. Only two layers are clearly distinguishable when poststained with uranyl acetate and lead citrate. The apical zone of dehiscence is characterized by a distinct annular swelling which appears during early ascosporogenesis. By spore maturation, this swelling is not evident either at the light or electron microscopic level. Instead, there appear to be both physical and chemical changes in the area of dehiscence. The wall is distinctly thinner and much more electron transparent in the area of dehiscence when treated with silver methanamine.

An Electron Microscopic Investigation of the Pathogenesis of Hemorrhage Induced by Rattlesnake Venom

1974 ◽  
Vol 32 ◽  
pp. 56-57
Author(s):  
Charlotte L. Ownby ◽  
Robert A. Kainer ◽  
Anthony T. Tu
Keyword(s):  
Phosphate Buffer ◽  
Osmium Tetroxide ◽  
Microscopic Investigation ◽  
Uranyl Acetate ◽  
Electron Microscopic Investigation ◽  
Lead Citrate ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Rattlesnake Venom ◽  
Diamondback Rattlesnake

One of the significant changes induced by the injection of rattlesnake (Crotalidae) venom is hemorrhage. Since crotaline antivenin does not prevent such local tissue damage, a more effective treatment of snakebite is needed. To aid in the development of such a treatment the pathogenesis of venom-induced hemorrhae was investigated.Swiss-Webster white mice were injected intramuscularly with Western diamondback rattlesnake (Crotalus atrox) venom. Two minutes after the injection, muscle tissue was obtained by bioosy from the thigh and fixed in 6% glutaraldehyde in Milloniq's phosphate buffer (DH 7.4, 2 hrs., 4°C). After post-fixation in 2% osmium tetroxide in Milloniq's phosphate buffer (pH 7.4, 1hr., 4°C) the tissue was dehydrated routinely in ethanol and embedded in Epon 812. The thin sections were stained with uranyl acetate in methanol and lead citrate then observed with either a Zeiss EM 9A or an Hitachi HS-8 electron microscope.

Immuno-Electron Microscopic Study on the Pulmonary Metastases of Chang' s Hepatoma Cells

1977 ◽  
Vol 35 ◽  
pp. 418-419
Author(s):  
E. C. Chew ◽  
C. N. Sun ◽  
H. J. White
Keyword(s):  
Tumor Metastasis ◽  
Pulmonary Metastases ◽  
Local Treatment ◽  
Hepatoma Cells ◽  
Uranyl Acetate ◽  
Lead Citrate ◽  
Electron Microscopic ◽  
Time Intervals ◽  
Pulmonary Vessels ◽  
Routine Procedures

The study of tumor metastasis has long been a subject of interest in cancer research because it is one of the principal problems in malignancy that prevents successful local treatment. The purpose of this communication is to report the sequence of metastasis of Chang's hepatoma cells studied by immuno-EM techniques.The use of peroxidase-labelled antifibrin antibody to demonstrate fibrin in early stages of metastases has been described earlier (1). Rats were killed at different time intervals, ranging from 5 minutes to 1 month after tumor cell innoculation. Small pieces of lung tissue were fixed in 4% phosphate buffered glutaraldehyde and post-fixed in 1% oxmium tetroxide. Routine procedures were followed for dehydration and embedding in Epon 812. Sections were stained with uranyl acetate and lead citrate.The fine structure of Chang's hepatoma cells has been reported elsewhere (2,3). At 5 minutes after IV injection, tumor cells were found in the pulmonary vessels.

Pores in the Plasma Membranes of Myocardial Cells

1967 ◽  
Vol 25 ◽  
pp. 198-199
Author(s):  
Glenwood P. Epling
Keyword(s):  
Electron Microscopy ◽  
Plasma Membranes ◽  
Polyester Resin ◽  
Uranyl Acetate ◽  
Lead Citrate ◽  
Myocardial Cells ◽  
Pinocytotic Vesicles ◽  
Sarcotubular System ◽  
The Right ◽  
Copper Mesh

Heart muscle samples were collected from the right ventricles of twelve mature cattle and twelve pigs at slaughter and immediately prepared for electron microscopy by immersion in three different fixatives: 40% OsO4 dissolved in CCl4, 1.5% OsO4 in S collidine buffer, and in 2.5%. glutaraldehyde followed by 1.5%, OsO4. Some tissue from each animal was processed in each fixative and was embedded in either an epoxy resin or in a polyester resin. Sections were cut at 300 to 600 Å in thickness on a thermally advancing microtome, using glass and diamond knives, and were mounted on copper mesh grids. Uranyl acetate and lead citrate stains were used. Tissues were examined in an RCA, EMU-3F electron microscope.In both the cattle and pigs, some heart samples contained myocardial cells with plasma membranes which invaginated into the cells to form tubular appearing channels. These channels occurred in addition to the transverse sarcotubular system, and in addition to the pinocytotic vesicles. The basement membrane which surrounds myocardial cells did not extend into the invaginations, but bridged over their external apertures.

Electron Microscopic Study of a Spontaneous Rat Tumor

1971 ◽  
Vol 29 ◽  
pp. 322-323
Author(s):  
P. W. Cole ◽  
R. M. Jamison
Keyword(s):  
Propylene Oxide ◽  
Uranyl Acetate ◽  
Female Rat ◽  
Lead Citrate ◽  
Sprague Dawley ◽  
Electron Microscopic ◽  
Non Invasive ◽  
Ultrathin Sections ◽  
Glass Knife ◽  
Rat Tumor

A spontaneously occurring, non-invasive mammary tumor was observed in a 7-month-old, non-lactating, random-bred Sprague-Dawley female rat. The tumor was located subcutaneously in the distal mammary line as a firmly encapsulated, lobated growth. The tumor was surgically removed and immersed in Clark's buffer containing 4% glutaraldehyde. (Later conventional histochemical studies revealed this tumor to be a relatively non-differentiated fibro-adenoma.) Exterior and interior portions of the tumor were excised and fixed separately. The tissues were cut into 1 mm cubes and fixed for 4 hours in 4% glutaraldehyde. The specimens were then washed 4 times in buffer and left overnight at 4°C in the buffer. The cubes were postfixed in 2% OsO4 for 2 hours, after which they were dehydrated in a graded series of ethanol. The specimens were then washed with 2 changes of propylene oxide and perfused with a 1:1 mixture of propylene oxide-Epon 812 for 1 hour. Next, the tissue cubes were embedded in a mixture of Epon 812, DDSA, NMA, and DMP 30. Polymerization was allowed to proceed sequentially at 37°C overnight, 45°C for 8 hours, and 60°C for 24 hours. Ultrathin sections were cut with the Porter-Blum MT-2B ultramicrotome equipped with a glass knife. Sections were picked up on copper grids and stained with saturated uranyl acetate and 0.2% lead citrate. Specimens were examined in the Philips 300 electron microscope at instrumental magnifications ranging from 3,000-20,000 times.

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