scholarly journals THE REACTIVITY AND STAINING OF TISSUE PROTEINS WITH PHOSPHOTUNGSTIC ACID

1969 ◽  
Vol 40 (3) ◽  
pp. 761-767 ◽  
Author(s):  
Lloyd Silverman ◽  
David Glick
Keyword(s):  
Phosphotungstic Acid ◽  
Negative Contrast ◽  
Mitochondrial Matrix ◽  
Quantitative Electron Microscopy ◽  
Thin Sections ◽  
Minimal Distortion ◽  
Fixation Treatment ◽  
Intense Electron ◽  
Positively Charged ◽  
Aqueous Acidic Medium

After aldehyde-fixation, treatment with phosphotungstic acid (PTA) in aqueous acidic medium was shown to produce an intense electron-opaque stain with minimal distortion of organelles. Mitochondrial matrix, cisternae of the endoplasmic reticulum, and the Z-band of muscle were densely stained, whereas membranes stood out in negative contrast. Staining of glycogen or lipid was not apparent. Under certain conditions the stain density reflected the concentration of protein based on the quantitative reaction of PTA with the positively charged groups, although the stoichiometry of the reaction between PTA and protein varied with the kind of protein. The staining conditions established should provide a base for the use of the method in quantitative electron microscopy, particularly on thin sections.

scholarly journals MEASUREMENT OF THICKNESS WITHIN SECTIONS BY QUANTITATIVE ELECTRON MICROSCOPY

1969 ◽  
Vol 40 (3) ◽  
pp. 768-772 ◽  
Author(s):  
Lloyd Silverman ◽  
Berit Schreiner ◽  
David Glick
Keyword(s):  
Electron Microscopy ◽  
Phosphotungstic Acid ◽  
Exchange Resin ◽  
Interference Microscopy ◽  
Quantitative Electron Microscopy ◽  
Thin Sections ◽  
Calculated Mass ◽  
Thickness Standard ◽  
Embedding Medium ◽  
Measurement Of Thickness

To apply the method of quantitative electron microscopy to the measurement of mass in thin sections, the thickness of the section at or very near the structure to be studied must be known. Dowex anion exchange resin AG 1 x 2, stained with phosphotungstic acid (PTA) at pH 6.4, was used as a thickness standard which could be embedded and sectioned. The sectioned PTA-Dowex appeared uniformly stained and exhibited suitable electron opacity. The stoichiometry of the reaction between PTA and the Dowex resin was measured by three independent methods based on gravimetric, colorimetric, and nitrogen determinations whose results showed close agreement. From the PTA uptake, the density of the stained spheres was calculated. Mass of a defined area of PTA-Dowex was measured by quantitative electron microscopy, and from this mass and density, the volume and then the thickness were calculated. The values for thickness were compared to those obtained by interference microscopy on the embedding medium alone in the same sections.

Ultrastructure of the adrenal cortex in frozen thin sections

1977 ◽  
Vol 27 (1) ◽  
pp. 303-311
Author(s):  
M.M. Magalhaes
Keyword(s):  
Adrenal Cortex ◽  
Lipid Droplets ◽  
Phosphotungstic Acid ◽  
Negative Contrast ◽  
Uranyl Acetate ◽  
Frozen Sections ◽  
Cell Organelles ◽  
Structural Localization ◽  
Thin Sections ◽  
Adrenal Cells

Pieces of both unfixed and fixed rat adrenal cortex were frozen and cut with a cryoultramicrotome. In unstained frozen sections of unfixed adrenals patches of heterochromatin were seen in the nucleus, while numerous round uniformly dense structures, the mitochondria, could be identified in the cytoplasm. Lipid droplets appear as small ligh round areas. Staining the sections with potassium permanganate, phosphotungstic acid, or uranyl acetate, or immersing them in glutaraldehyde, increased the contrast of cell organelles. In frozen sections of fixed material, membranes became visible in negative contrast. Mitochondria display vesicular cristae, whereas the Golgi complex, lysosomes and peroxisomes are easily recognizable. The possibility of cutting frozen sections of unfixed material with reasonable morphological preservation allows the fine-structural localization of steroids in adrenal cells after applying autoradiographic emulsion to the sections.

Membranes in lupin root nodules. II. Preparation and properties of peribacteroid membranes and bacteroid envelope inner membranes from developing lupin nodules

1978 ◽  
Vol 30 (1) ◽  
pp. 151-174
Author(s):  
J.G. Robertson ◽  
M.P. Warburton ◽  
P. Lyttleton ◽  
A.M. Fordyce ◽  
S. Bullivant
Keyword(s):  
Sucrose Gradient ◽  
Phosphotungstic Acid ◽  
Nadh Oxidase ◽  
Osmotic Shock ◽  
Root Nodules ◽  
Freeze Fracture ◽  
Electrophoretic Analysis ◽  
Thin Sections ◽  
Peribacteroid Space ◽  
Gradient Fractionation

Peribacteroid membranes and bacteroid envelope inner membranes have been isolated from developing lupin nodules. Isolation of the peribacteroid membranes was achieved by first preparing membrane-enclosed bacteroids free from other plant organelles or membranes. The peribacteroid membranes were then released by osmotic shock and purified by centrifugation to equilibrium on sucrose gradients. The bacteroids were broken in a pressure cell and the bacteroid envelope inner membranes were isolated using sucrose gradient fractionation of the bacteroid total envelope preparation. The density of the peribacteroid membranes decreased during the period of development of N2-fixation in lupin nodules from 1.148 g/ml for nodules from 12-day plants to 1.137 g/ml for nodules from 18-day plants. The density of the bacteroid envelope inner membranes from nodules from 18-day plants was 1–153 g/ml. The identity and homogeneity of the isolated membranes was established, by comparison with membranes in intact nodules, using phosphotungstic acid and silver staining of thin sections and particle densitites on faces of freeze-fracture replicas of the membranes. Analyses for NADH oxidase and succinate dehydrogenase, spectral analyses and gel-electrophoretic analysis of proteins were also used to characterize the membrane and soluble protein fractions from the nodules. The ratio of lipid to protein was 6.1 for the peribacteroid membranes and 2.5 for the bacteroid envelope inner membranes. Leghaemoglobin was localized in the plant cytoplasm in lupin nodules and not in the peribacteroid space.

scholarly journals ON PHOSPHOTUNGSTIC ACID STAINING. I

1971 ◽  
Vol 19 (11) ◽  
pp. 641-647 ◽  
Author(s):  
G. QUINTARELLI ◽  
R. ZITO ◽  
J. A. CIFONELLI
Keyword(s):  
Heavy Metal ◽  
Sialic Acid ◽  
Chondroitin Sulfate ◽  
Phosphotungstic Acid ◽  
Model Systems ◽  
Organic Cations ◽  
Hydroxyl Groups ◽  
Sublingual Gland ◽  
Ph Dependent ◽  
Positively Charged

In this study histochemical experiments have been carried out in order to understand the "staining mechanism" of phosphotungstic acid (PTA). One of the main objectives of this project was to investigate the mode of interaction of the heavy metal and to define the type of functional groups in the substrates responsible for PTA binding. Therefore, tissues containing known macromolecules were selected and utilized as model systems. Epiphyseal cartilage, rat sublingual glands, human bone and purified collagen were used to study the interaction of the polyacid with chondroitin sulfate (cartilage), sialic acid (sublingual gland) and collagen (purified collagen and bone). The results obtained suggested that PTA does not interact with chondroitin sulfate, with sialic acid or with the hydroxyl groups of the sugar moieties of these macromolecules. Rather, the binding appeared to be selective for positively charged groups. Since PTA interaction to organic cations was pH-dependent, it is suggested that the heavy metal binds by means of coulombic forces and that no hydrogen bonds are involved.

scholarly journals Electron microscope study of reassociation of spectrin and actin with the human erythrocyte membrane

1981 ◽  
Vol 90 (1) ◽  
pp. 70-77 ◽  
Author(s):  
S Tsukita ◽  
S Tsukita ◽  
H Ishikawa ◽  
S Sato ◽  
M Nakao
Keyword(s):  
Erythrocyte Membrane ◽  
Human Erythrocyte ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Erythrocyte Membranes ◽  
Human Erythrocyte Membrane ◽  
Thin Sections ◽  
Fixation Treatment ◽  
Human Erythrocyte Membranes ◽  
Inside Out

Reassociation of spectrin and actin with human erythrocyte membranes was studied by stereoscopic electron microscopy of thin sections combined with tannic acid- glutaraldehyde fixation. Treatment of the erythrocyte membrane with 0.1 mM EDTA (pH 8.0) extracted more than 90 percent of the spectrin and actin and concomitantly removed filamentous meshworks underlying the membranes, followed by fragmentation into small inside-out vesicles. When such spectrin-depleted vesicles were incubated with the EDTA extract (crude spectrin), a filamentous meshwork, similar to those of the original membranes, was reformed on the cytoplasmic surface of the vesicles. The filamentous components, with a uniform thickness of 9 nm, took a tortuous course and joined one another often in an end-to-end fashion to form a irregular but continuous meshwork parallel to the membrane. Purified spectrin was also reassociated with the vesicles in a population density of filamentous components almost comparable to that of the crude spectrin-reassociated vesicles. However, the meshwork formation was much smaller in extent, showing many independent filamentous components closely applied to the vesicle surface. When muscle G-actin was added to the crude spectrin- or purified spectrin- reassociated vesicles under conditions which favor actin polymerization, actin filaments were seen to attach to the vesicles through the filamentous components. Two modes of association of actin filaments with the membrane were seen: end-to-membrane and side-to- membrane associations. In the end-to-membrane association, each actin filament was bound with several filamentous components exhibiting a spiderlike configuration, which was considered to be the unit of the filamentous meshwork of the original erythrocyte membrane.

scholarly journals MEASUREMENT OF PROTEIN CONCENTRATION BY QUANTITATIVE ELECTRON MICROSCOPY

1969 ◽  
Vol 40 (3) ◽  
pp. 773-778 ◽  
Author(s):  
Lloyd Silverman ◽  
David Glick
Keyword(s):  
Electron Microscopy ◽  
Protein Concentration ◽  
Clinical Laboratory ◽  
Red Cells ◽  
Cell Protein ◽  
Red Cell ◽  
Protein Loss ◽  
Quantitative Electron Microscopy ◽  
Thin Sections ◽  
Mean Values

The method of quantitative electron microscopy was applied to the measurement of protein concentration in thin sections. The human erythrocyte was selected as a model because of its apparently uniform protein concentration. Phosphotungstic acid (PTA) in aqueous solution was used as a reversible stain for protein, and PTA-stained Dowex resin spheres were embedded along with the red cells as standards for measurement of section thickness. The mass of stain removed from a given area of sectioned red cell by buffer (pH 7.4) was measured by quantitative electron microscopy. From the stoichiometry of the reaction between PTA and red cell protein established in this study, the amount of protein present in the measured area was calculated. From this amount of protein and the measured thickness, the concentration of protein was calculated and expressed as g/100 ml, for comparison with the clinical laboratory value for hemoglobin. Groups of red cells from the same sample were measured on 3 different days and their mean values (g/100 ml ± SD) were 29 ± 3.9, 30 ± 2.7, and 33 ± 4.6, compared to the clinical laboratory value of 32.1 g/100 ml packed cells, after correction for volume change and protein loss during fixation.

A rapid phosphotungstic acid staining method on ultra-thin sections

1994 ◽  
Vol 52 ◽  
pp. 318-319
Author(s):  
K. Chien ◽  
R. L. Van de Velde ◽  
R.C. Heusser ◽  
H. Shiroishi ◽  
A.H. Cohen
Keyword(s):  
Room Temperature ◽  
Phosphotungstic Acid ◽  
Staining Method ◽  
Tap Water ◽  
Petri Dish ◽  
Hereditary Disease ◽  
Microwave Oven ◽  
Staining Procedure ◽  
Thin Sections ◽  
Nail Patella Syndrome

The Nail-Patella Syndrome (NPS) is an autosomal dominant hereditary disease in which about 60% of the patients have glomerular abnormalities. Phosphotungstic acid (PTA) has been used successfully to demonstrate the specific collagen fibrils of NPS within the glomerular basement membrane and/or mesangial matrix in ultra-thin sections. However, in our laboratory we had difficulty staining Eponate 12 sections with PTA. A modified staining procedure which makes use of the microwave has been developed as follows:1. Glutaraldehyde/osmium fixed kidney specimens were embedded in Eponate 12 and ultra-thin sections collected on Gilder 400 mesh copper grids. Grids were stained using a RTV 700 silicone rubber staining pad. This pad contains shallow, round wells (5mm in diameter and 2mm deep) with slots in the center bottom of each well. By bending the pad, the rim of a grid can be inserted into the slot. On release, the grids adhere securely to the wall of the slot.2. 10% aqueous phosphotungstic acid was dropped into the staining wells around each grid. The staining pad was then placed in a moisture chamber consisting of a pre-heated Petri dish containing wetted filter paper and placed in Pelco 3400 laboratory microwave oven for 110 seconds at 80% power. A Tripour beaker containing 200ml room temperature tap water was placed in the microwave oven as a heat sink.

The organization of the microtubule associated protein tau in Alzheimer paired helical filaments

1993 ◽  
Vol 51 ◽  
pp. 190-191
Author(s):  
George C. Ruben ◽  
Khalid Iqbal ◽  
Inge Grundke-Iqbal ◽  
John E. Johnson
Keyword(s):  
Paired Helical Filaments ◽  
Brain Lesions ◽  
Alzheimer Type ◽  
Thin Sections ◽  
Protein Tau ◽  
Transmission Electron ◽  
Normal Individuals ◽  
Thin Sectioning ◽  
Fixation Treatment ◽  
10 Nm Filaments

Neurofibrillary tangles (NFT) of paired helical filaments (PHF) are the most characteristic brain lesions of Alzheimer disease and their abundance determines the diagnosis. This histopathological hallmark occurs with greater frequency in patients with clinical dementia of the Alzheimer type than in normal individuals of the same age. Therefore, it is important that we establish and understand the arrangement of subunits in PHF. This knowledge, we believe, could be key to understanding the pathogenesis of neurofibrillary degeneration.Intracellular Alzheimer NFT fixed in OsO4 or KMnO4 in the absence of glutaraldehyde treatment were first observed by transmission electron microscopy (TEM) of thin sections and reported by Kidd. The tangles he observed contained helical pairs of ~10 nm filaments separated center to center by 15 nm with a double helical period of ~160 nm that he named paired helical filaments. Subsequent thin sectioning work with a similar fixation treatment confirmed that the PHF were helical with crossover width, T=12.8±2 nm, and a wide region, W=24±3.1 nm, which occured every L=65−80 nm.

A Transmission Electron Microscopy Study and Viability Evaluation of the Effect of 2450 MHz Microwaves on Candida albicans.

1997 ◽  
Vol 3 (S2) ◽  
pp. 117-118
Author(s):  
C. Melgoza ◽  
J. Sepúlveda ◽  
M. Moreno ◽  
M. A. Rodríguez
Keyword(s):  
Microscopy Study ◽  
Uranyl Acetate ◽  
Electron Microscopy Study ◽  
Microwave Oven ◽  
Yeast Cells ◽  
Growth Media ◽  
Mitochondrial Matrix ◽  
Transmission Electron Microscopy Study ◽  
Thin Sections ◽  
Transmission Electron

Microwaves of 2450 MHz have been used as a heating procedure in food technology, tissue processing for histological studies (1), in medicine (2) and as a sterilization agent for medical devices (3). Experiments were carried out exposing yeast cells of Candida albicans suspended in growth media to different times of radiation at 750 watts in a commercial microwave oven. Batches of 5 tubes containing the cells suspension were radiated from 15 seconds to 3 minutes and colony counts were done after exposure. Viability ceased totally after one minute radiation. Samples were obtained from unexposed controls and tubes radiated for 15, 30, 45 and 60 seconds, centrifuged and the pellets fixed in 1.5% potasium permanganate. After embbeding in Epon, thin sections were obtained and stained with uranyl acetate. Micrographs were taken with a Zeiss EM-109 electron microscope. Cells showed alteration of internal membranes at early times, and later changes indicative of cytoplasmic coagulation and organelle disorganization were seen. At 15 seconds mitochondrial matrix showed alterations followed by damage of its membranes.

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