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PubMed1983

Identification of dictyosomelike structures (DLS) isolated from spermatocyte-enriched fractions of guinea pig testes.

Dictyosomelike structures (DLS) are organelles unique to male mammalian germ cells and occur in large numbers in spermatocytes of the guinea pig. Selective staining of DLS with phosphotungstic acid at low pH was used to identify DLS in cell fractions prepared by combined sucrose gradient and differential centrifugation procedures. This staining property was retained in homogenates and permitted DLS to be distinguished from cisternae of Golgi apparatus and most other spermatocyte membranes. DLS were resistant to the homogenization and isolation stresses, at least in regard to their structural integrity. The purity of the DLS fractions was proportional to the purity of spermatocytes derived in the initial cell separation steps. Cell separation and the attainment of good spermatocyte fractions appear critical to the isolation of DLS by these procedures.

Animals↗
PubMed1990

Screening of myocardial contraction bands: a comparison between two histological staining methods.

Two histological stains for demonstration of contraction bands (cb): Mallory's phosphotungstic acid hematoxylin (PTAH) and a modification of luxol fast blue (LFB) were compared. Microscopical sections from hearts from 60 autopsies were screened. The regional distribution of the cb lesions was also studied. It was found that PTAH showed cb more consistently than LFB. None of the stains were suitable for screening in the lowest magnification. In cases of cardiac death the cb were often extensive and scattered throughout the myocardium. In non-cardiac deaths with cb these were always discrete and seen only in one location which might be a means of differentiating lesions of pathogenetic importance from agonal ones.

Aged↗
PubMed1994

Long-term effects of intermittent versus continuous ethanol exposure on hippocampal synapses of the rat.

The hippocampus is known to be very sensitive to a large spectrum of different neurotoxins including ethanol. Ethanol administered continuously or intermittently may affect the hippocampus in different ways. Intermittent administration of ethanol has many features in common with the low level electrical stimulation protocols which lead to the functional changes associated with the phenomenon of kindling. In this study, the differential effects of intermittent intraperitoneal ethanol injections (3 g/kg twice daily) and continuously administered ethanol in drinking water (20%) on hippocampal synapses in the rat were studied using ethanolic phosphotungstic acid staining and electron microscopy. After 1 month of intermittent exposure a significant reduction (18%) of synapses was seen in the stratum lucidum of the CA3 region. Continuously treated animals showed no significant change over this time despite a higher total ethanol intake. In the dentate gyrus, a compensatory increase in supragranular synaptic number was seen only in continuously treated animals. These findings demonstrate the sensitivity of synapses of the hippocampus to the presence of ethanol and the larger effects of peaking ethanol concentrations compared to more constant levels. These results emphasize the need to consider the differential effects of various types of ethanol consumption also on the human brain.

Animals↗
PubMed1998

Immunohistochemical, ultrastructural and molecular study of well differentiated adenocarcinomas of the lung predominantly composed of goblet cells.

In order to clarify the morphological and biological characteristics of well differentiated adenocarcinoma of the lung predominantly composed of goblet cells (WDAG), histopathological examinations, including some molecular biological procedures, were carried out using 42 surgical specimens of primary lung carcinoma which were predominantly (>50% of the total cell population) or totally composed of goblet cells. The subjects included 19 men and 21 women, ranging in age from 41 to 81 (mean 60 years old) with predominantly nodular, peripherally located lesions. Ultrastructural examination revealed characteristic apical microvillous filamentous core rootless (AFCR) in some, but not all, cases. Histologically, these AFCR corresponded well with structures stained by phosphotungstic acid hematoxylin (PTAH). The goblet cells of WDAG were divided into PTAH-positive (26 cases) and -negative (16 cases) groups. The PTAH-positive group had larger tumor size, greater number of intrapulmonary and extrapulmonary metastases and shorter disease-free interval. The immunoexpression of p53 protein (60%) and rate of K-ras point mutation (84%) were also higher in the PTAH-positive group. Therefore the goblet cell population of WDAG, though it may appear morphologically homogeneous under light microscopy, is actually composed of heterogeneous groups of cells with different histopathological characteristics and biological behavior.

Adenocarcinoma↗
PubMed1992

Immunocytochemical demonstration of dopamine-beta-hydroxylase and cytochrome B561 on the axonal reticulum in bovine sympathetic neurons.

In sympathetic neurons the axonal reticulum can be considered an extension of the secretory pole of the Golgi apparatus. If this tubular system indeed represents the neurosecretory apparatus, it would likely contain on its membranes the enzymes involved in catecholamine synthesis. To test this hypothesis, we investigated the distribution of dopamine-beta-hydroxylase and cytochrome b561 in bovine splenic nerve and nerve terminals in the vas deferens with an immunogold procedure after glycolmethacrylate embedding. Counterstaining with phosphotungstic acid at low pH selectively revealed the axonal reticulum elements. With antibodies against both enzymes, gold labeling was observed over the large dense-cored vesicles, the Golgi-associated axonal reticulum, the reticulum within axons, and the tubular complex at the nerve terminal. From our results it can be concluded that in sympathetic neurons the axonal reticulum represents a tubular neurosecretory system, extending from the Golgi apparatus in the cell soma to the nerve terminal. This concept emphasizes the local production of neurosecretory vesicles and may be of importance in the interpretation of neuronal transmission in normal and diseased states.

Animals↗
PubMed2002

Ultrastructural analyses of the Caenorhabditis elegans DR 847 bli-1(n361) mutant which produces abnormal cuticle blisters.

The bli-1 gene of Caenorhabditis elegans has been previously described as a mutation which disrupts the structure of the adult-stage cuticle causing the formation of fluid-filled blisters. We investigated the blistering phenotype exhibiting n361 allele and observed a gradual blister formation in adult nematodes. In the course several fine changes occurred including a high electron density granulous material filling the intermediate layer, alterations on struts structure, and finally the total disappearance of the fibrous and basal layers. With the ethanolic phosphotungstic acid technique (E-PTA), which localizes basic proteins, reaction product was observed in the cortical layer of the wild strain, whereas in the mutant strain an irregular labelling pattern was observed in this region. The granulous material inside the intermediate layer of the mutant strain showed also a strong reaction. An imidazole-buffered osmium tetroxide solution was used to visualize lipids at ultrastructural level, however no dense product was detected in the cuticle of both strains of C. elegans. Based on these results we postulated that the blistering phenotype is due to an altered function of bli-1 gene, which is probably enzymatic.

Animals↗
PubMed1983

Metal compound intensification of the electron-density of diaminobenzidine.

Diaminobenzidine (DAB), commonly used in immunocytochemistry as the substrate for peroxidase, has a low electron density. DAB has a known affinity for the salts of some metals and therefore an examination of the ability of six metal compounds (including osmium tetroxide) to increase the electron density associated with DAB deposits has been undertaken. Ultra-thin sections of unosmicated rat pituitary gland, embedded in L. R. White resin, were immunostained by a hapten sandwich immunoperoxidase method, using antibodies to ACTH and TSH. The unintensified electron density of the DAB polymer reaction product on the specific endocrine granules was compared with the electron density resulting from the use of each of the six metal compounds. Lead and silver nitrate gave unsatisfactory results, while phosphotungstic acid and uranyl acetate produced a limited increase in specific electron density under the conditions used. Gold chloride was found to give the highest electron density to the specific endocrine granules, followed closely by osmium tetroxide. Background staining was greater when osmium was used. We conclude that several metal compounds may be used to intensify the electron density of DAB, but of the ones tested, gold chloride, which is safer, more stable, and cheaper than osmium tetroxide, was clearly the best. This approach not only increases the electron density of the DAB reaction product, but allows of the possibility of quantitation using energy dispersive X-ray analysis.

3,3'-Diaminobenzidine↗
PubMed1969

Measurement of thickness within sections by quantitative electron microscopy.

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.

Colorimetry↗
PubMed1998

Glycation changes the charge distribution of type I collagen fibrils.

In aging and diabetes, glycation of collagen molecules leads to the formation of cross-links that could alter the surface charge on collagen fibrils, and hence affect the properties and correct functioning of a number of tissues. The electron-optical stain phosphotungstic acid (PTA) binds to positively charged amino acid side-chains and leads to the characteristic banding pattern of collagen seen in the electron microscope; any change in the charge on these side-chains brought about by glycation will affect the uptake of PTA. We found that, upon glycation, a decrease in stain uptake was observed at up to five regions along the collagen D-period; the greatest decrease in stain uptake was apparent at the c1 band. This reduction in PTA uptake indicates that the binding of fructose leads to an alteration in the surface charge at several sites along the D-period. Not all lysine and arginine residues are involved; there appear to be specific residues that suffer a loss of positive charge.

Age Factors↗
PubMed1979

E-PTA stains oligodendroglial surface membranes and microtubules in optic nerves during myelination.

Aldehyde fixed Xenopus tadpole and frog optic nerves were stained en bloc with ethanolic phosphotungstic acid (E-PTA). During rapid myelination, intense staining was observed on cytoplasmic faces of paranodal terminal loops and loosely wrapped oligodendroglial membranes found along inner and outer surfaces of compact myelin sheaths. Oligodendroglial microtubules also were heavily stained. Where stained cytoplasmic faces fused to form a lamella of compact myelin, the intense staining was reduced to a thinner, fainter line. In optic nerves of adult frogs, the staining was less dense but the pattern was similar. The staining distribution and available histochemical evidence indicate that E-PTA stains positively charged proteins non specifically. Since myelin basic protein is found in oligodendroglia during myelination, we suggest that it is being stained by E-PTA while being transported along microtubules to sites where it is inserted into developing myelin lamellae.

Animals↗
PubMed1979

Cytochemical studies of ameloblasts and the surface layer of enamel of the rat incisor at the maturation stage.

In order to elucidate the cytochemical properties of the membranous structure between enamel and ameloblasts of the rat incisor at the maturation stage, chromic phosphotungstic acid (Cr-PTA) and periodic acid-silver methenamine (PA-silver) techniques for electron microscopy were employed in combination with a digestion test with hyaluronidase, neuraminidase, collagenase or trypsin. Also, acid phosphatase activity of ameloblasts at the maturation stage was examined with a modified GOMORI's metal salt method. An intensely Cr-PTA reactive band approximately 0.1 micron thick appeared along the surface layer of enamel at the transitional stage, and at the very beginning of the maturation stage another intensely Cr-PTA reactive band which was seen by uran-lead stain to be a delicate electron-dense membranous structure appeared as well between enamel and ameloblasts. A lot of cytoplasmic small vesicles or tubular structures, both intensely reactive to Cr-PTA, were observed near the apical membranes of the overlying ameloblasts indicating that those organelles must have been responsible for the secretion of the latter band. Acid phosphatase activity was clearly demonstrated at Cr-PTA reactive large vesicles in the cytoplasm of those cells. The PA-silver staining technique manifested a band heavily deposited with silver grains along the surface layer of enamel, i.e., where the former band existed, but showed no particular reaction at the latter, the band-like layer between enamel and ameloblasts. Hyaluronidase or neuraminidase treatment remarkably decreased the Cr-PTA reaction of the latter band. Trypsin or collagenase treatment, on the other hand, not only eliminated the Cr-PTA reaction but digested the band itself. These results suggest that the membranous structure between enamel and ameloblasts of a rat incisor is not so-called enamel cuticle but a basal lamina produced by overlying ameloblasts and that the basal lamina contains collagenous components even though it lies on enamel.

Ameloblasts↗
PubMed1991

Ultrastructural localization of basic lysine-rich proteins during the nucleologenesis in preimplantation mouse embryos.

The paper describes nucleologenesis in preimplantation mouse embryos studied by electron microscopy after staining with ethanolic phosphotungstic acid (E-PTA), a substance which binds to basic lysine-rich proteins and makes it thus possible to follow their localization and distribution. A nucleolar precursor body (NPB) homogeneously stained with E-PTA first appears in pronuclei of a fertilized oocyte. In 2-cell stage embryos, the staining intensity of the central part of NPB is reduced, whereas the outer zone retains the PTA positivity. In the course of further development, reticulation takes place in the peripheral zone and all structures of a functional nucleolus are gradually formed. Tiny spherical bodies, called "remnant NPBs", can still be observed in reticular nucleoli of cells at the morula stage. The similarities in reaction and morphological appearance of the NPB and the corresponding structures in oocytes of the mouse and rat antral follicle are discussed.

Animals↗
PubMed1990

Quantifying synaptic number and structure: effects of stain and post-mortem delay.

Current research indicates the importance of synaptic number and structure in plastic processes such as development, learning and memory, and aging. As such, the examination of these neural features has become an important factor in research on human conditions such as mental retardation, aging and Alzheimer's disease. Synaptic research in human tissue typically involves delayed post-mortem fixation, therefore the current research was designed to examine the effect of post-mortem delay on synaptic number and structure in tissue stained with either routine osmium lead citrate/uranyl acetate (osmium) or ethanol phosphotungstic acid (EPTA). Results indicate that synaptic density shows either a gradual decline (EPTA) or an initial marked drop followed by a plateau (osmium) up to 10-15 h post-mortem depending on the stain used. The number of synaptic vesicles per synapse also undergoes a gradual decline. Measures of synaptic structure were more stable, with the primary change being an initial increase in the cross-sectional length of the synapse. Maximal height of the pre- and postsynaptic dense elements were not affected by post-mortem delay. The EPTA stain gave the best estimates of synaptic parameters with short post-mortem delays. These results indicate that different synaptic measures (and stains) show different responses to post-mortem fixation delay, and that experimental or statistical methods must be used to control for post-mortem effects.

Animals↗
PubMed2002

Morphometry of E-PTA stained synapses at the periphery of pathological lesions.

We carried out a novel application of the disector sampling and counting method, in a biopsy material from the pathologic human brain, to estimate the synaptic structural dynamics, quantitatively. Parietal cortex biopsies of adult (mean age: 40.0 years) and old (mean age: 66.2 years) patients having undergone surgical intervention were investigated. The tissue samples were excised at the periphery of meningioma masses. Synaptic contact zones were stained en bloc by the ethanol phosphotungstic acid (E-PTA) preferential technique which selectively enhances both the pre- and post-synaptic paramembranous material separated by a sharp cleft against a very faint background, thus facilitating and objectifying synaptic morphometry. The disector method, associated with currently used morphometric formulas, enabled us to measure the number of synapses/m3 of tissue (numeric density: Nv); the total area of the synaptic contact zones/m3 of tissue (surface density: Sv) and the average synaptic size (S). In old vs. adult patients, Nv decreased by 7.5% (Mean (SEM): Adult 2.0040(0.0452); Old 1.6780(0.0623)), while S increased by 17.5% (Adult 0.0203(0.0026); Old 0.0246(0.0035)). Sv did not show any age-related difference. The same negative correlation between Nv and S has also been reported in physiological aging, and this suggests the active presence of age-related synaptic restructuring mechanisms in the nervous tissue surrounding a tumoral mass.

Adult↗
PubMed1989

Multilayered distribution of peptidoglycan in the periplasmic space of Escherichia coli.

When a staining technique using phosphotungstic acid (PTA) in 10% (w/v) chromic acid was applied to cells of Escherichia coli, the periplasmic space was seen as a dark 15-nm-thick layer of uniform appearance and constant width. Our observations are consistent with peptidoglycan being the main material stained. Isolated sacculi as well as purified peptidoglycan (protein free) were also stained by the same procedure, the thickness of the peptidoglycan being 8.8 +/- 1.8 and 6.6 +/- 1.5 nm, respectively. The increased thickness of the PTA-stained layer in stationary phase cells correlated well with the increased thickness of isolated sacculi or purified peptidoglycan and with the increased amount of peptidoglycan in such cells. Thickness measurements on isolated peptidoglycan were compatible with a two to three layer structure for material from exponential phase cells and with a four to five layer structure for that from stationary phase cells. Furthermore, the results indicated an uneven distribution of peptidoglycan material in the periplasmic space, the peptidoglycan spanning the space from the inner to the outer membrane.

Chromates↗
PubMed2002

Application of an automated specimen search system installed in a transmission electron microscope for the detection of caliciviruses in clinical specimens.

To evaluate the performance of an automated specimen search system in the detection of caliciviruses such as Norwalk-like viruses and Sapporo-like viruses, a suitable negative staining method was developed and the viruses were examined using the system installed in a transmission electron microscope (TEM). Clear images of the viruses were obtained by staining with 2% uranyl acetate at pH 4.0 as compared with 2% phosphotungstic acid staining at any pH. When the image parameter of 30+/-6 nm for the diameter of a single virus-like particle of 2% uranyl-acetate-stained Norwalk-like virus was set on the automated specimen search system, 95% of the virus-like particles that were counted by the conventional TEM technique were detected. The system was used to detect Norwalk-like viruses in five semipurified stool samples in which Norwalk-like viruses had already been detected by reverse transcription-polymerase chain reaction assay and conventional electron microscopy. The positive detection rate for Norwalk-like viruses, which had been counted by the conventional technique, ranged from 56.2 to 77.9% using this system. Our findings indicate that the automated specimen search system installed in a TEM is suitable for the detection of caliciviruses in semipurified stool samples. The system is useful for clinical diagnosis without the need for operator intervention.

Automation↗
PubMed1969

Measurement of protein concentration by quantitative electron microscopy.

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.

Blood Proteins↗
PubMed1987

A simple staining method for chromatin in electron microscopy compatible with serial sectioning.

The study of the disposition of chromatin in the interphasic nucleus requires the combination of serial sectioning and a specific or preferential chromatin staining. The staining of chromatin with phosphotungstic acid (PTA) chromatin was originally employed on sections of glycolmethacrylate-embedded samples. As it is very difficult to obtain ribbons with this resin, we introduced a modification which consisted of staining the tissue after fixation and before dehydration, in order that epoxy resins can be applied. Several procedures were tried and the best results were attained in the following way. Standard fixation of samples no thicker than 1 mm was carried out with 2.5% glutaraldehyde at pH 7.2 for 1 or 2 h at room temperature; tissues were then rinsed three times with 0.2N HCl adjusted to pH 2.1-2.3 with 0.2N NaOH for 15 min. Staining was held with 3% W/V PTA in 1N HCl adjusted to the same pH. Samples were dehydrated in gradual ethyl alcohol concentrations and Epon-embedded. Post-staining on sections with uranyl-acetate and lead-citrate or other methods may be used to demonstrate other cell components and their relations to chromatin.

Animals↗