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PubMed1982

Dense bodies in silver-stained spermatocytes of the Chinese hamster: behavior and cytochemical nature.

From the silver staining behavior of various organelles in the nucleus we have divided meiotic prophase (leptotene to the diffuse stage) of the male Chinese hamster into five stages. Components within the nucleus, such as synaptonemal complex (SC), sex bivalent (SB), nucleolus organizer regions (NORs), chromatin and the dense bodies, showed a characteristic feature in each stage of meiotic prophase. The lampbrush chromosome stage was found to be followed by the diffuse stage. The chromatin around SC began to be organized at early pachytene and formed a brush-like structure at late pachytene. During early prophase stages a dramatic change in SB morphology occurred. Three types of morphology of SB were recognized: (1) the XY pair with long synapsis and fusiform or diffuse thickening of the unpaired portions (late zygotene and early pachytene), (2) desynapsed, branched, and anastomosed axes seen at late pachytene. Two types of the dense body were found during meiotic prophase; the double body in early stage (leptotene to early pachytene) and the single body in later stages (mid pachytene to diffuse stage). The small precursors of the double body existed at early leptotene but they increased in size and also changed the silver stainability during zygotene, becoming the characteristic double body consisted of one light body (L-body) and one dark body (D-body). These two bodies can also be recognized after Giemsa or acridine orange (AO) staining. The L-body fluoresced reddish orange after AO staining. The single body, which is probably formed by amalgamation of the D- and the L-bodies, showed a staining reaction similar to that of the D-body. Data from pancreatic lipase and protease treatments suggest that the D-body contained a lipoprotein.

Animals↗
PubMed1975

The pattern of ocular dominance columns in macaque visual cortex revealed by a reduced silver stain.

A pattern of alternating dark and pale bands was observed in the striate cortex of the macaque monkey. The bands, which ran parallel to the surface, were seen in tangential sections stained with a reduced silver method for normal fibers and were most clear in layer 4C alpha, immediately deep to the line of Gennari. The dark bands were about 300 mu wide and showed blind endings and bifurcations. The light bands were about 50 mu wide and did not branch or terminate within area 17. Because the dark bands were similar in width to the bands of terminal degeneration which have been shown to result from single-layer lesions of the lateral geniculate body, it seemed possible that they corresponded to ocular dominance columns. To test this idea, the boundaries of ocular dominance columns were marked in a physiological experiment: tangential electrode penetrations were made in an anesthetized monkey and, as the electrode was advanced horizontally in the fourth layer, the eye preference of single units and of the background activity was monitored. Small electrolytic lesions were placed at the points where a change in eye preference occurred. The brain was subsequently fixed, sectioned tangentially and stained with the silver method. All the lesions--total of 12 --fell directly on the pale bands. Moreover, the electrode had not passed over any pale bands without a lesion being placed. It was concluded that the dark bands do correspond to single ocular dominance columns and the pale bands to the boundaries between columns. The banding appearance is due to a greater density of tangential fibers within columns than at the borders of columns. These tangential fibers are in part the preterminal arborizations of geniculocortical axons, since some of them have been shown to degenerate after geniculate lesions. The ocular dominance columns were mapped for most of the striate cortex, using serial tangential sections stained with the silver method. The overall pattern was ,imilar in several monkeys, though the details of the branching arrangements varied from animal to animal. The columns met the 17-18 border at right angles. On the outer surface of the hemisphere the columns converged from the 17-18 border, turned medially with repeated fusions of columns, and streamed over the lip of the calcarine fissure. In the roof of the fissure they met a second system of columns oriented parasagittally. In terms of the visual field, the columns ran roughly horizontally for the central 10 degrees of the field, and circumferentially beyond that. The columns were not mapped in the stem of the fissure, the area corresponding to the far periphery of the field. The constancy of column width across the cortex probably allows a functional matching between ocular-dominance and orientation columns.

Animals↗
PubMed1988

Polyacrylamide gel electrophoresis and silver staining for detection of rotavirus in stools from diarrheic patients in Thailand.

Detection of rotavirus by polyacrylamide gel electrophoresis in combination with silver staining and by enzyme-linked immunosorbent assay showed 96.7% identical results in tests with 1,304 stool specimens from diarrheic patients. The polyacrylamide gel electrophoresis method can be modified to reduce cost and working time. Phenol extraction of stools, however, is essential in maintaining the sensitivity of the method.

Diarrhea↗
PubMed2001

Correlation between silver-stained nucleolar organizer region area and cell cycle time.

BACKGROUND: The relationship between the population doubling time and the quantity of silver-stained nucleolar organizer region (AgNOR) interphase proteins was studied in cell culture at three different temperatures used to modulate the cell cycle duration. METHODS: After MIB 1 and AgNOR combined staining, the quantity of AgNOR proteins was measured in cycling cells by image cytometry. RESULTS: Among the several parameters calculated, the AgNOR relative area showed a strong correlation with the changes of the population doubling time induced by different temperatures. CONCLUSIONS: The results support the hypothesis that the cell cycle time and the size of the ribogenesis machinery are coregulated and that measurements of AgNORs can thus be used as a static evaluation of the cell cycle duration in arbitrary units.

Antigens, Nuclear↗
PubMed1986

Silver-stained nucleolar organizer regions in bone marrow cells and peripheral blood lymphocytes of Philadelphia chromosome-positive chronic myelocytic leukemia patients.

Silver-stained nucleolar organizer regions (Ag-NOR) in bone marrow cells and/or phytohemagglutinin-stimulated peripheral blood lymphocytes were compared between six normal healthy persons as controls and 22 Philadelphia chromosome (Ph)-positive chronic myelocytic leukemia (CML) patients, to examine if any disease associated changes occur in the expression of Ag-NOR. Although the frequency of Ag-NOR-positive cells and the number of Ag-NOR per cell were generally greater in lymphocytes than in bone marrow cells in both controls and CML patients, the Ag-stainability of these cell types in CML patients was considerably heterogeneous, compared with that found in controls. The peripheral lymphocytes of CML patients in the chronic phase, but not in the blastic phase, exhibited a significantly lowered Ag-stainability when compared with those of controls. while no such difference was observed between bone marrow cells of controls and leukemia patients in both phases of CML. In the blastic phase, however, the occurrence of Ag-NOR on the Ph of CML bone marrow cells was significantly less than expected. The present findings are discussed in relation to the existing data on the Ag-NOR expression in both normal and neoplastic cells.

Adolescent↗
PubMedDate not supplied

MK-801 neurotoxicity in cupric silver-stained sections: lesion reconstruction by 3-dimensional computer image analysis.

Routine histopathologic evaluation of the brain (paraffin embedding, hematoxylin and eosin staining) makes it difficult for an investigator to identify the overall location and relative extent of lesions as they relate to neural substructures. Moreover, it is very difficult to convey this information to others who are less familiar with neuroanatomy. This study combined a 3-dimensional imaging program with a cupric silver stain for neuronal degeneration in order to determine the location and extent of a focal lesion produced by MK-801 (dizocilpine maleate), a glutamate receptor antagonist that induces necrosis in a small population of neurons in the cortex of rats. A male Sprague-Dawley rat was treated with a subcutaneous dose of MK-801 (10 mg/kg) and was perfused with fixative through the left ventricle 3 days after treatment, a time point known to reveal maximal neurotoxic effects. The brain was embedded in a gelatin matrix, frozen, and serially sectioned at a thickness of 40 microm. The cupric silver method of de Olmos was used to stain frozen sections at 320-microm intervals. Using a color charged-couple device (CCD) camera and a macro lens, a series of 2-dimensional images, which encompassed the entire rostral to caudal extent of the brain, was captured. A computer program was written to define internal and external boundaries in these 2-dimensional images. Then, 3-dimensional reconstructions were generated on a Silicon Graphics workstation using IRIS "Explorer." The quality of the 3-dimensional reconstructions allowed for easy identification of various neural substructures while clearly revealing the exact location and extent of the resulting necrotic neurons that were positively identified by the cupric silver stain. This 3-dimensional lesion reconstruction method provides a powerful tool for conveying spatial information about the nature of neurotoxic lesions in the brain. In addition, it may be used to investigate further dose-response relationships and the effects of other neurotoxicants.

Animals↗
PubMed1986

[Nature of the silver staining and association of acrocentric chromosomes in normal and leukemic human cells].

Silver staining and acrocentric chromosome association (ACA) patterns were investigated in bone marrow cells as well as in peripheral blood cells (cultures with or without PHA) from 39 patients with chronic myelocytic leukemia (CML), including 20 cases being in blastic phase (BP CML), and 51 patients with acute leukemia (AL). Bone marrow cells and PHA-stimulated peripheral blood lymphocytes (from 10 and 17 healthy donors, respectively) were used as a control. The frequency of ACA in metaphases from bone marrow cells of all the above groups of patients was shown to be decreased compared to that in PHA-stimulated lymphocytes. Patients with BP CML and AL constituted the most heterogeneous groups although some of them demonstrated the highest ACA-frequency per cell. There is a pronounced correlation between Ag+-nucleolus organizer regions (NOR's) and the frequency of ACA. With the exception of CML, the correlation coefficients (0.83, 0.74, and 0.72) were highly significant for all the above groups (donors, BP CML, and AL patients, respectively). The distribution pattern of single chromosome pairs, according to their ACA frequency, differed with every individual studied, but it was similar in normal and leukemic cells of the same individual. From the above data a conclusion is made that the frequency of ACA may depend on the functional activity of the NOR's as well as on the cells type.

Acute Disease↗
PubMed1994

The use of immuno-gold silver staining in bluetongue virus adsorption and neutralisation studies.

The immuno-gold-silver staining (IGSS) technique was used in scanning electron microscopy for the detection and semi-quantitation of low copy antigens on the surface of cells. The methodology was exploited in experiments designed to examine the interaction of small numbers of virus particles with the surface of susceptible host cells. Using bluetongue virus (BTV) as an example, IGSS procedures confirmed that maximum adsorption occurred within 60 min and that adsorbed virus particles were distributed randomly on the surface of the cell. Neutralising antibody did not prevent binding of BTV to the plasma membrane, but abrogated virus uptake. The use of IGSS in the study of virus-cell interactions was validated by transmission electron microscopy and classical biochemical experiments utilising radioactively-labelled virus.

Adsorption↗
PubMed1995

Location of focal silver staining at endothelial gaps in inflamed venules examined by scanning electron microscopy.

The century-old histological technique of silver nitrate staining has proven to be extremely useful for visualizing endothelial cell borders and localizing endothelial gaps, but the significance of the staining is still not fully understood. To gain some insight into what silver nitrate stains, we developed a method that enabled us to use scanning electron microscopy with backscattered and secondary electron imaging to examine silver staining at endothelial cell borders of venules of the rat tracheal mucosa. We found that in normal venules, silver lines followed the smooth contour of cell borders. However, 1 min after endothelial permeability was increased by substance P, cell borders were irregular and displaced from the silver lines by as much as 4.3 microns, and the lines were accompanied by three types of silver deposits. Most common (46% of total) were annulus-shaped silver deposits that surrounded endothelial gaps. These deposits averaged 1.5 microns in width, were positioned symmetrically across cell borders, and were located at a depth of 0.3 micron beneath the luminal surface. Many endothelial gaps were partitioned into multiple pores (mean, 2.4) by fingerlike processes of endothelial cells. Surprisingly, the gaps occupied only 5.4% of the total area of the silver deposits and constituted 0.15% of the luminal surface of the leaky postcapillary venules. A second type of silver deposit (19% of total) was positioned asymmetrically with respect to the cell border and marked sites where endothelial cell margins still overlapped but appeared to be vertically separated by obliquely oriented gaps. A third type marked gaps at three-cell junctions; these were no more abundant than deposits elsewhere around the cell perimeter, suggesting that three-cell junctions were not unusually leaky sites. We conclude that silver nitrate marks endothelial cell borders and outlines endothelial cell gaps by staining an element of intercellular junctions. The annular shape of many silver deposits around gaps suggests that junctional elements in the apposing cells are separated during gap formation but are still present at the gap perimeter.

Animals↗
PubMed1984

Two-dimensional electrophoresis and "ultrasensitive" silver staining of cerebrospinal fluid proteins in neurological diseases.

Cerebrospinal fluid (CSF) proteins, as resolved by two-dimensional electrophoresis and made visible by silver staining, have been examined in patients with various neurological diseases and normal volunteers. The patterns for 15 of 20 patients with Parkinson's disease showed a protein (Mr 25 000) with charge similar to albumin, which was not seen in the patterns for any of 91 normal volunteers. Patterns for 21 of 22 multiple sclerosis patients showed novel immunoglobulin light chain proteins, also not present in the CSF of any normal volunteers. Quantitative analysis by computer-assisted densitometry in Parkinson's disease and multiple sclerosis showed that 20 of 68 and 33 of 85 proteins, respectively, were significantly altered as compared with proteins in the normal population. This ability to characterize both Parkinson's disease and multiple sclerosis molecularly provides a broad baseline for improved clinical diagnosis and may serve as an aid in exploring the underlying pathophysiology. These studies illustrate the potential of applying this methodology in the study of neurological diseases.

Adult↗
PubMed1994

[Histological grading of ductal pancreatic carcinomas. Relationship to silver staining of NOR-associated proteins and to p53 immunoreactivity].

The histological grading of 29 ductal pancreatic carcinomas was compared with that of silver stained NOR-associated proteins and with p53 immunoreactivity. The AgNOR number (NZ) and AgNOR areas (NF) of 10 grade I carcinomas, 13 grade II carcinomas and 6 grade III carcinomas were investigated by image analysis. Furthermore, the protein expression of the p53 gene was examined by immunohistochemistry. A significant difference was found in both AgNOR parameters between grade I, grade II and grade III carcinomas (NZ: p < 0.01; NF: p < 0.05; grade I carcinomas: NZ 5.1/NF 8.2 microns 2; grade II carcinomas: NZ 6.0/NF 11.3 microns 2 grade III carcinomas: NZ 8.4/NF 15.2 microns 2). By contrast no relation was found between histological grading and p53 immunoreactivity. In 56% of all carcinomas positive evidence was found of the p53 gene product. (6 grade I carcinomas, 7 grade II carcinomas, 3 grade III carcinomas). 44% of the cases were p53-negative (4 grade I carcinomas, 6 grade II carcinomas, 3 grade III carcinomas). This study demonstrates that the AgNOR technique is useful in the grading of ductal pancreatic carcinomas, whereas the expression of the p53 gene product is not.

Carcinoma, Ductal, Breast↗
PubMed1987

Electrophoresis of honey: characterization of trace proteins from a complex biological matrix by silver staining.

The proteins of unconcentrated honey have been detected with the methylamine-incorporating silver stain (T. Marshall, 1984, Anal. Biochem. 136, 340-346) following sodium dodecyl sulfate-polyacrylamide gel electrophoresis or high-resolution two-dimensional electrophoresis. The former consistently reveals at least 19 protein bands in a variety of Australian honeys. Two-dimensional electrophoresis gave patterns of poor resolution but proved useful for further characterization of the major protein constituents. The protein patterns were unaffected by centrifugation of the samples prior to preparation for electrophoresis.

Electrophoresis, Polyacrylamide Gel↗
PubMed1994

Silver stained nucleolar organizer region proteins and Helix pomatia agglutinin immunostaining in esophageal carcinoma: correlated prognostic factors.

The number of nucleolar organizer region proteins identified by silver staining (AgNORs) and alterations in cell surface glycoproteins identified by Helix pomatia agglutinin (HPA) immunostaining were studied in 96 primary esophageal carcinomas. The number of AgNORs increased with increasing depth of cancer invasion, venous involvement, lymphatic invasion, and tumor stage. Positivity of HPA staining correlated significantly with increasing depth of cancer invasion, venous invasion, and tumor stage. Forty of 42 HPA negative cases had low AgNORs numbers (< 4), while 39 of 54 HPA positive cases had high AgNORs numbers (> or = 4) (P = 0.0001). The survival rate of patients with stage III and IV disease was significantly poorer for those with a high AgNORs score and HPA positivity than in those with a low AgNORs score and HPA negativity. The present study indicates that the AgNORs score is positively correlated with positive HPA staining and that tumors having both factors represent a subgroup of esophageal carcinomas with poor prognosis.

Adult↗
PubMed1992

Relationship between quantity of silver stained nucleolar organizer regions associated proteins (Ag-NORs) and population doubling time in ten breast cancer cell lines.

Using a one-step silver staining technique ten human breast cancer cell lines were investigated, determining by means of automated image analysis the mean area of Ag-NORs per nucleus, the total number of Ag-NORs, and the so-called "scattered" and "clustered" Ag-NORs. These parameters were statistically correlated with the population doubling time (PDT) of the various carcinoma cell lines of this series. Our results show that the mean area of Ag-NORs per nucleus (p less than 0.001) is highly significantly correlated to the PDT, whereas all other parameters were less or not significant. It is concluded that the assessment of the area of Ag-NORs by automated image analysis is a simple and reliable method for the determination of cell duplication rates.

Breast Neoplasms↗
PubMed1985

An immunogold silver staining method for the light microscopic analysis of blood lymphocyte subsets with monoclonal antibodies.

This paper introduces an immunogold silver staining procedure for the identification of human lymphocyte subpopulations in optical microscopy. The described procedure is performed on cells in suspension (peripheral mononuclear cells or blood buffy-coat cells) and uses conventional antilymphocytic monoclonal antibodies (OKT3, OKT4, OKT8, OKIa1) followed by gold-labeled secondary antibodies (GAM G30 or G40) and silver sensitization. It ends with the preparation of permanent records (smears or cytocentrifuge preparations) which are counterstained with standard panoptic Wright or May-Grünwald Giemsa stain. In all cases, the surface antigens appear as numerous black dots on the lymphocytes, with a strong labeling reaction that allows one to clearly distinguish between negative and positive cells. The comparison with immunofluorescence microscopy in normal individuals and in patients indicates that this new immunostaining technic is specific and more sensitive. It has the advantages of using small amounts of blood (1 mL per test), of being rapid (three hours), and of allowing the simultaneous immunophenotypic and morphologic evaluation on smears. This makes it suitable for the analysis of other biologic specimens not already accessible with immunofluorescence. The labeled preparations apparently can be stored indefinitely, and are thus useful for longitudinal studies in the same patient. Finally, the method does not require sophisticated equipment and may be subjected to automated analysis.

Antibodies, Monoclonal↗
PubMed1998

G protein gamma subunits coimmunoprecipitated with antibodies against alpha subunits: identification of major isoforms in cultured cells by silver stain and immunoblotting with conventional transfer procedure.

The betagamma subunits of G proteins were coimmunoprecipitated with antibodies against various alpha subunits, and analyzed by silver stain and immunoblotting with conventional transfer procedure and membrane-blocking buffer containing 2% BSA. Multiple isoforms of gamma were coimmunoprecipitated with no significant difference in form or ratio among the antibodies against alpha subunits used, suggesting antibodies against any alpha subunit could coimmunoprecipitate all forms of gamma. Therefore, this method was applicable to analyze gamma subunits in various cells, especially to clarify what forms of gamma subunits are major components. The major isoforms were: gamma5 in C6, NG108-15, HeLa, HEK293, and F9 cells; gamma12 in Swiss 3T3 and BRL-3A cells; and gamma3 in PC12 cells. In addition to most gamma subunits identified, unidentified gamma subunits were present in PC12, NG108-15, and BRL-3A cells. Furthermore, the method was applied to examine changes of isoforms of gamma during differentiation of HL-60 cells. Undifferentiated cells mainly contained gamma5, but retinoic acid treatment of cells replaced most gamma5 with gamma2. Thus, this method is useful to determine the major isoforms which seem to be the more important in cells.

Amino Acid Sequence↗
PubMed2001

A new silver staining apparatus and procedure for matrix-assisted laser desorption/ionization-time of flight analysis of proteins after two-dimensional electrophoresis.

We report on a new silver stain especially developed for staining large gels (25 cm x 20 cm) from the Hoefer ISO-DALT system for matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis of proteins. The staining protocol can be summarized as follows: the gels are sensitised in tetrathionate/potassium acetate solution and washed several times in distilled water. After impregnation with silver nitrate, the silver is reduced in the presence of potassium carbonate, thiosulphate and formaldehyde. The staining procedure is stopped with Tris/acetate after which the gels are rinsed and stored in water before spot picking for MALDI-TOF analysis is performed. This protocol has several advantages over existing ones. The gels are stained in a new apparatus that reduces gel handling to a minimum thus also reducing the contamination with keratins to a minimum. The development times in potassium carbonate are very long (up to 40 min) thus improving batch-to-batch reproducibility. Only the surface of the proteins is stained and the silver can be oxidized, thereafter MALDI-TOF can be performed with protein loads as little as 100 micrograms per gel.

Cell Line↗