An enhanced cellular organelle visualization procedure when staining for peroxisomes

1995 ◽  
Vol 53 ◽  
pp. 886-887
Author(s):  
J. W. Horn ◽  
B.J. Dovey-Hartman
Keyword(s):  
Tissue Architecture ◽  
Ultrastructural Examination ◽  
Fixed Tissue ◽  
Tissue Samples ◽  
Tissue Sections ◽  
Visual Identification ◽  
Transmission Electron ◽  
Light Staining ◽  
Cellular Components

Reliable visual identification of peroxisomes is important in developmental, clinical, and investigational research. The current technique employed in most laboratories uses a specific electron dense label for the demonstration of peroxisomes by transmission electron microscopy by applying 3,3'- Diaminobenzidine Tetrahydrochloride (DAB) directly to freshly fixed tissue samples to react with endogenous peroxisomal catalase. After routine processing, ultrastructural examination of tissue sections is conducted either with light staining or without post-staining of grids. While peroxisomes are easily identified using this method, remaining tissue architecture is difficult to visualize due to the opacity of the tissue. Additionally, if grids are post-stained with heavy metal solutions, they must be modified to allow for enough staining to visualize cellular components without compromising the quality of the peroxisome label. We will describe a technique whereby DAB-reacted tissues are stained with a postfixative solution including potassium ferricyanide that imparts density to cell membranes and cellular components thereby enhancing identification and interpretion of data.

Discovery of intracellular 2,8 dihydroxyadenine crystals in bovine lymphatic and hepatic cells

1987 ◽  
Vol 45 ◽  
pp. 906-907
Author(s):  
W. E. Rigsby ◽  
D. M. Hinton ◽  
V. J. Hurst ◽  
P. C. McCaskey
Keyword(s):  
Polarized Light ◽  
Paraffin Sections ◽  
Tissue Samples ◽  
Whole Cells ◽  
Tissue Sections ◽  
Hepatic Cells ◽  
Slaughter House ◽  
Mammalian Tissues ◽  
Carbon Coated ◽  
Cellular Components

Crystalline intracellular inclusions are rarely seen in mammalian tissues and are often difficult to positively identify. Lymph node and liver tissue samples were obtained from two cows which had been rejected at the slaughter house due to the abnormal appearance of these organs in the animals. The samples were fixed in formaldehyde and some of the fixed material was embedded in paraffin. Examination of the paraffin sections with polarized light microscopy revealed the presence of numerous crystals in both hepatic and lymph tissue sections. Tissue sections were then deparaffinized in xylene, mounted, carbon coated, and examined in a Phillips 505T SEM equipped with a Tracor Northern X-ray Energy Dispersive Spectroscopy (EDS) system. Crystals were obscured by cellular components and membranes so that EDS spectra were only obtainable from whole cells. Tissue samples which had been fixed but not paraffin-embedded were dehydrated, embedded in Spurrs plastic, and sectioned.

scholarly journals The Application of Paraphenylenediamine Staining for Assessment of Phospholipidosis

2016 ◽  
Vol 44 (8) ◽  
pp. 1160-1165 ◽  
Author(s):  
Norimitsu Shirai ◽  
Frank J. Geoly ◽  
Walter F. Bobrowski ◽  
Carlin Okerberg
Keyword(s):  
Study Drug ◽  
Tissue Preparation ◽  
Lamellar Bodies ◽  
Intracellular Accumulation ◽  
Drug Induced ◽  
Tissue Samples ◽  
Tissue Sections ◽  
Unsaturated Lipids ◽  
Transmission Electron ◽  
Fine Granular Material

Drug-induced phospholipidosis is characterized by intracellular accumulation of phospholipids with lamellar bodies in cells exposed to xenobiotics. Demonstration of the lamellar bodies by transmission electron microscopy (TEM) is the hallmark for a definitive diagnosis of phospholipidosis. However, the preparation of tissue samples for TEM and their ultrastructural evaluation are technically challenging and time consuming. Paraphenylenediamine (PPD) is essentially a fat stain, and the staining mechanism is based upon the osmication of unsaturated lipids. Thus, the application of PPD staining to osmicated tissue samples is considered an optimal way to identify lipids. We evaluated the potential of PPD staining to localize phospholipid accumulations on osmium-fixed semi-thin tissue sections of the lung, kidney, and liver, which were affected with phospholipidosis, under a light microscope. PPD staining revealed the presence of PPD positive dark fine granular material in the cytoplasm for all affected tissues examined, which correlated ultrastructurally with lamellar bodies as well as a light microscopic finding of cytoplasmic vacuolation. The great advantage of PPD is that it can be incorporated into the protocol for standard TEM tissue preparation and significantly improve the efficiency of TEM work. In conclusion, PPD provides a simple, sensitive, and reliable method for visualizing phospholipid accumulation on light microscopy and represents an easy tool to study drug-induced phospholipidosis.

scholarly journals Reversible embedment cytochemistry (REC): a versatile method for the ultrastructural analysis and affinity labeling of tissue sections.

1986 ◽  
Vol 34 (2) ◽  
pp. 177-188 ◽  
Author(s):  
G Gorbsky ◽  
G G Borisy
Keyword(s):  
Electron Microscopy ◽  
Cultured Cells ◽  
Cell Structure ◽  
Linear Polymers ◽  
Fixed Tissue ◽  
Affinity Labels ◽  
Tissue Sections ◽  
Culture Cells ◽  
Transmission Electron ◽  
Critical Point Drying

Reversible embedment cytochemistry (REC) is a new method for revealing cellular ultrastructure and for improving access of intracellular targets to macromolecular affinity labels. Fully polymerized polymethylmethacrylate was dissolved in dichloromethane and infiltrated into fixed tissue-culture cells and tissues. After evaporation of the solvent, samples were left in hard plastic. Samples were thus embedded without exposure to chemical polymerization reactions that might damage tissue ultrastructure or antigenicity. Glass or diamond knives fitted with water troughs were used to cut sections 30-1000 nm thick. Since polymethylmethacrylate is composed of linear polymers that are not covalently crosslinked, the plastic was easily extracted from the sections by immersion in solvent. Subsequently, various preparative methods, including negative staining, critical point-drying, and platinum-carbon rotary shadowing, were used to provide detailed images of well-preserved cell structure for conventional and high-voltage transmission electron microscopy. Fluorescein-conjugated affinity labels were used to obtain subcellular distributions of target molecules in semi-thick sections of cultured cells and tissues for light microscopy. Colloidal gold-labeled antibodies were used to localize microtubules in sections of cultured cells by electron microscopy. REC is a versatile method that should find wide application in many studies of cellular function.

Simultaneous Localization and Quantitation of Progesterone Receptor Immunoreactivity in Human Breast Carcinomas.

1997 ◽  
Vol 3 (S2) ◽  
pp. 7-8
Author(s):  
K.A. Roth
Keyword(s):  
Image Analysis ◽  
Progesterone Receptor ◽  
Reaction Products ◽  
Breast Carcinomas ◽  
Human Breast ◽  
Fixed Tissue ◽  
Tissue Samples ◽  
Tissue Sections ◽  
Human Breast Carcinomas ◽  
Estrogen And Progesterone Receptor

The presence of estrogen and progesterone receptors on human breast carcinomas correlates with both response to hormonal therapy and survival. Quantitation of receptor activity is typically performed by radioreceptor assay on fresh frozen tumor tissue obtained at the time of surgical resection. Estrogen and progesterone receptor reactivity can also be detected immunohistochemically on frozen sections of breast cancer and in appropriately fixed paraffin embedded tissues. Several studies have shown that semi-quantitative visual determination and quantitative image analysis assessment of estrogen and progesterone receptor immunoreactivity on fixed tissue sections is highly correlated with biochemical measurements of receptor in frozen samples. We recently developed a procedure for the simultaneous localization and quantitation of antigens in fixed tissue samples that does not involve antigen extraction, radioactive materials, or image analysis. In this procedure, which we term a “Midwestern assay”, fixed tissue sections are used with antigen-specific enzyme-linked antibodies to generate soluble reaction products which are spectrophotometrically quantitatable, as in an ELISA, and deposited reaction products which are microscopically localizable, as in enzyme-based immunohistochemistry.

Observability of Very Thick Specimen by Using Transmission Scanning Electron Microscope

1971 ◽  
Vol 29 ◽  
pp. 26-27
Author(s):  
K. Shibatomi ◽  
T. Yamanoto ◽  
H. Koike
Keyword(s):  
Electron Microscope ◽  
Image Quality ◽  
Scanning Electron Microscope ◽  
Chromatic Aberration ◽  
Image Contrast ◽  
Objective Lens ◽  
Thick Specimen ◽  
Transmission Electron ◽  
Scanning Electron

In the observation of a thick specimen by means of a transmission electron microscope, the intensity of electrons passing through the objective lens aperture is greatly reduced. So that the image is almost invisible. In addition to this fact, it have been reported that a chromatic aberration causes the deterioration of the image contrast rather than that of the resolution. The scanning electron microscope is, however, capable of electrically amplifying the signal of the decreasing intensity, and also free from a chromatic aberration so that the deterioration of the image contrast due to the aberration can be prevented. The electrical improvement of the image quality can be carried out by using the fascionating features of the SEM, that is, the amplification of a weak in-put signal forming the image and the descriminating action of the heigh level signal of the background. This paper reports some of the experimental results about the thickness dependence of the observability and quality of the image in the case of the transmission SEM.

The annealed (0001) α-alumina surface and its influence on thin-film growth

1995 ◽  
Vol 53 ◽  
pp. 336-337
Author(s):  
Michael W. Bench ◽  
Paul G. Kotula ◽  
C. Barry Carter
Keyword(s):  
Electron Microscopy ◽  
Surface Energy ◽  
Film Growth ◽  
Thin Film Growth ◽  
Energy Calculations ◽  
Transmission Electron ◽  
Reflection Electron Microscopy ◽  
Low Energy Electron ◽  
Surface Nature

The growth of semiconductors, superconductors, metals, and other insulators has been investigated using alumina substrates in a variety of orientations. The surface state of the alumina (for example surface reconstruction and step nature) can be expected to affect the growth nature and quality of the epilayers. As such, the surface nature has been studied using a number of techniques including low energy electron diffraction (LEED), reflection electron microscopy (REM), transmission electron microscopy (TEM), molecular dynamics computer simulations, and also by theoretical surface energy calculations. In the (0001) orientation, the bulk alumina lattice can be thought of as a layered structure with A1-A1-O stacking. This gives three possible terminations of the bulk alumina lattice, with theoretical surface energy calculations suggesting that termination should occur between the Al layers. Thus, the lattice often has been described as being made up of layers of (Al-O-Al) unit stacking sequences. There is a 180° rotation in the surface symmetry of successive layers and a total of six layers are required to form the alumina unit cell.

Microprobe analysis of inclusion bodies related to two benzofuran derivatives

1987 ◽  
Vol 45 ◽  
pp. 672-673
Author(s):  
T. L. Benning ◽  
P. Ingram ◽  
J. D. Shelburne
Keyword(s):  
Inclusion Bodies ◽  
Uranyl Acetate ◽  
Multiple Organ ◽  
High Dose ◽  
En Bloc ◽  
Tissue Samples ◽  
Transmission Electron ◽  
Organ Systems ◽  
Dense Inclusion ◽  
Melanin Complex

Two benzofuran derivatives, chlorpromazine and amiodarone, are known to produce inclusion bodies in human tissues. Prolonged high dose chlorpromazine therapy causes hyperpigmentation of the skin with electron-dense inclusion bodies present in dermal histiocytes and endothelial cells ultrastructurally. The nature of the deposits is not known although a drug-melanin complex has been hypothesized. Amiodarone may also cause cutaneous hyperpigmentation and lamellar lysosomal inclusion bodies have been demonstrated within the cells of multiple organ systems. These lamellar bodies are believed to be the product of an amiodarone-induced phospholipid storage disorder. We performed transmission electron microscopy (TEM) and energy dispersive x-ray microanalysis (EDXA) on tissue samples from patients treated with these drugs, attempting to detect the sulfur atom of chlorpromazine and the iodine atom of amiodarone within their respective inclusion bodies.A skin biopsy from a patient with hyperpigmentation due to prolonged chlorpromazine therapy was fixed in 4% glutaraldehyde and processed without osmium tetroxide or en bloc uranyl acetate for Epon embedding.

Methods to Monitor and Improve the Performance of Specimen Holders for Transmission Electron Cryomicroscopy

1996 ◽  
Vol 54 ◽  
pp. 454-455
Author(s):  
B. L. Armbruster ◽  
B. Kraus ◽  
M. Pan
Keyword(s):  
Electron Microscopy ◽  
Electron Beam ◽  
Electron Beam Irradiation ◽  
Beam Irradiation ◽  
Quality Of Data ◽  
Electron Cryomicroscopy ◽  
Thermal Equilibration ◽  
Transmission Electron ◽  
Electron Microscopes

One goal in electron microscopy of biological specimens is to improve the quality of data to equal the resolution capabilities of modem transmission electron microscopes. Radiation damage and beam- induced movement caused by charging of the sample, low image contrast at high resolution, and sensitivity to external vibration and drift in side entry specimen holders limit the effective resolution one can achieve. Several methods have been developed to address these limitations: cryomethods are widely employed to preserve and stabilize specimens against some of the adverse effects of the vacuum and electron beam irradiation, spot-scan imaging reduces charging and associated beam-induced movement, and energy-filtered imaging removes the “fog” caused by inelastic scattering of electrons which is particularly pronounced in thick specimens.Although most cryoholders can easily achieve a 3.4Å resolution specification, information perpendicular to the goniometer axis may be degraded due to vibration. Absolute drift after mechanical and thermal equilibration as well as drift after movement of a holder may cause loss of resolution in any direction.

A combined pseudoreplica-immunocytochemical technique for research and diagnostic virology

1990 ◽  
Vol 48 (3) ◽  
pp. 900-901
Author(s):  
Blayne Fritz ◽  
Stanley J. Naides ◽  
Kenneth Moore
Keyword(s):  
Phosphotungstic Acid ◽  
Immunogold Labelling ◽  
Uranyl Acetate ◽  
Distilled Water ◽  
Clinical Specimens ◽  
Tissue Sections ◽  
Specimen Retrieval ◽  
Transmission Electron ◽  
Viral Particles ◽  
Diagnostic Virology

The pseudoreplica method of staining viral particles for visualization by transmission electron microscopy is a very popular technique. The ability to concentrate clinical specimens while semi-embedding viral particles makes it especially well suited for morphologic and diagnostic virology. Immunolabelling viral particles with colloidal gold is a technique frequently employed by both research and diagnostic virologists. We have characterized a procedure which provides the advantage of both by modifying and combining pseudoreplica staining and immunogold labelling.Modification of specimen retrieval and delay of staining allows us to utilize pseudoreplica processed specimens within our standard immunogold labelling protocol. In brief, we absorbed samples onto 2% agarose, added.25% Formvar and wicked dry. We then floated the Formvar-virus film onto double distilled water, added grids and retrieved with parafilm. The Formvar-virus specimens were then treated as thin tissue sections within our standard two stage immunolabelling protocol. Following completion of immunogold labelling; each grid was negatively stained with phosphotungstic acid or uranyl acetate contrast stains.

Hepatocyte ultrastructural alterations in perimetastatic areas

1996 ◽  
Vol 54 ◽  
pp. 768-769
Author(s):  
H. J. Finol ◽  
M. E. Correa ◽  
L.A. Sosa ◽  
A. Márquez ◽  
N.L. Díaz
Keyword(s):  
Malignant Tumor ◽  
Malignant Tumors ◽  
Surrounding Tissue ◽  
Ultrastructural Changes ◽  
Pancreas Carcinoma ◽  
Duct Carcinoma ◽  
Tissue Samples ◽  
Primary Malignant Tumor ◽  
Transmission Electron ◽  
Remote Sites

In classical oncological literature two mechanisms for tissue aggression in patients with cancer have been described. The first is the progressive invasion, infiltration and destruction of tissues surrounding primary malignant tumor or their metastases; the other includes alterations produced in remote sites that are not directly affected by any focus of disease, the so called paraneoplastic phenomenon. The non-invaded tissue which surrounds a primary malignant tumor or its metastases has been usually considered a normal tissue . In this work we describe the ultrastructural changes observed in hepatocytes located next to metastases from diverse malignant tumors.Hepatic biopsies were obtained surgically in patients with different malignant tumors which metatastized in liver. Biopsies included tumor mass, the zone of macroscopic contact between the tumor and the surrounding tissue, and the tissue adjacent to the tumor but outside the macroscopic area of infiltration. The patients (n = 5), 36–75 years old, presented different tumors including rhabdomyosarcoma, leiomyosarcoma, pancreas carcinoma, biliar duct carcinoma and colon carcinoma. Tissue samples were processed with routine techniques for transmission electron microscopy and observed in a Hitachi H-500 electron microscope.

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