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PubMed1992

Modified silver staining for immobilized pH gradients.

Silver development of gels containing an immobilized pH gradient has proved difficult so far because the bonded buffers (especially the tertiary amino acrylamido derivatives) tend to absorb silver ions with a resultant heavy background of increasing darkness from the anode to the cathode. We report a variant of silver staining in which thiosulfate is used twice: (i) prior to silver impregnation, at the millimolar level, to enhance sensitivity, and (ii) during development, at the micromolar level, to decrease the background.

Hydrogen-Ion Concentration↗
PubMed1979

Silver-stained structures in mammalian meiotic prophase.

Silver staining of mammalian spermatocytes revealed, in light microscopy, synaptonemal complex and structures within the sex vesicle. It is feasible to follow the chromosome pairing phenomenon from zygotene to pachytene by examining the behavior of synaptonemal complexes. Nucleolus organizer regions take heavy silver stain in pachytene but are no longer detectable in later stages of meiosis.

Animals↗
PubMed2003

[Improvement of the silver-stain and it's test and verification in comet assay].

In order to find out a better silver-stain method so that to replace EB-stain in comet assay, the selected silver-stain methods were tested via determining fixing, coloring, displaying, fading, and testing the pigmentation temperature. When it was optimized, it was used in testing the DNA damage by two types of compounds, namely potassium dichromate (K2Cr2O7) and 1,2-dichloroethane (DCE) to two kind of cells, namely spleen cells of mouse and human outer lymphocyte, and, the sensitivity was compared with that of EB-stain. The results showed that the modified silver-stain was the first successful non-fluorescent dye method in comet assay that can dye clearly, permanently, with a light background and good contrast, and it was easy to manipulate and it was cheap. The stain can detect the DNA damage by compounds to cells and has the same positive threshold like that in EB-stain. It can even dye the small fragments that EB-stain cannot. It is suggested that in comet assay, the silver-stain can substitute the EB-stain completely.

Comet Assay↗
PubMed1984

A method for combined C-banding and silver staining.

A reliable technique for combined C-banding and silver staining of metaphase chromosomes which uses trypsinization is described. Slides are first immersed in dilute HCl to remove residual cytoplasm from around the chromosomes. They are then treated with saturated barium hydroxide and incubated overnight in saline sodium citrate (0.30 M NaCl, 0.03 M sodium citrate, adjusted to pH 7.0 with HCl). Following the C-banding pretreatment, a two-step method of silver staining which employs a protective colloidal developer is used to stain the nucleolar organizer regions (NORs) of the chromosomes. Silver staining is followed by trypsinization to remove extraneous silver precipitate from the chromosome arms which permits the C-bands to be stained with Giemsa. The method works equally well with fresh and aged mitotic chromosome preparations and gives consistent staining of both heterochromatin and active NORs in metaphases across the slide.

Chromosome Banding↗
PubMed1996

The distribution of Ag-NORs proteins and ribosomal RNA genes in human spermatogonia: an original procedure associating silver-staining, fish and confocal microscopy.

It is generally accepted that Ag-NORs proteins are associated with nucleolar components engaged in rRNA synthesis. Since conflicting results have been reported concerning the intranucleolar sites of active ribosomal genes, the relationships between the silver-stained areas and the distribution of rDNA were analyzed in human spermatogonia using confocal microscopy of fluorescent in situ hybridized preparations previously stained with silver. The fluorescent signal strictly corresponded to the silver-stained area, identified at the ultrastructural level as the fibrillar center and the surrounding dense fibrillar component. A quantitative evaluation of nucleolar activity solely based on silver-staining may be misleading, taking into account that only Ag-positive proteins located in the dense fibrillar component can be associated with transcriptional activity, while those stored in the fibrillar center are associated with non-transcribed rRNA genes.

DNA, Ribosomal↗
PubMed1996

A longitudinal study of human age-related ribosomal RNA gene activity as detected by silver-stained NORs.

The relative frequencies of silver-stained nucleolar organizing regions (Ag-NORs) as a function of age have been analyzed in skin fibroblasts derived from eight adult individuals participating in the Gerontology Research Center (GRO) Longitudinal Study, NIA, Baltimore, MD. Since silver staining of NORs is correlated with rRNA gene activity, we used this cytological method to examine the pattern of rRNA gene activity in specific individuals, each at two different ages. Our results show that the average number of Ag-NORs/cell decreases significantly with advancing age of each individual, presumably indicating a general pattern of age-related decline/alteration in rRNA gene activity and this pattern is individual-specific. The findings of our longitudinal study is consistent with the results of previous cross-sectional (population) studies on rRNA gene activity as detected by Ag-NORs. However, it appears that the relative rate in the age-related decline of rRNA gene activity, as evidenced by lower Ag-NOR frequencies with age, is variable from person to person.

Adult↗
PubMed1991

The observation of endothelial cells in vein grafts by the en face silver staining method.

The introduction of en face silver staining to microsurgery has provided useful, detailed information about endothelial cells with regard to their size, shape, and number. With this staining method, endothelial cells of rat vein grafts have been morphologically investigated following division of the site of observation into three areas: the recipient artery, the proximal part of the vein graft, and the midportion of the vein graft. The results show that the arterial endothelial cells are small and spindle-shaped, whereas the cells in the proximal vein graft location are larger. Finally, large, rounded endothelial cells are seen in the midportion of the graft. These results are quantitated through a computerized graphic analysis system, which provides estimations of the cell size, Feret's diameters, and irregularities in the cell borders. The combination of en face silver staining and computerized graphic analysis has been especially useful for comparing minute changes that occur in recovering/regenerating endothelial cells.

Animals↗
PubMed1994

Technical and methodological aspects of silver staining and measurement of nucleolar organizer region (NOR).

In the last few years the NOR silver-staining technique has been introduced in tumor pathology for both diagnostic and prognostic purposes, but the lack of a standardized staining protocol has frequently led to misinterpretation of actual structures evaluated by the various authors. Recent data have demonstrated that AgNOR area is closely related to the whole stained nucleolar area obtained by prolonging the silver staining reaction beyond the optimal time for selective NOR visualization. In order to compare data from different laboratories, we propose to stain the whole nucleolus by silver and to measure the dimensions of the silver-stained nucleoli by image analysis.

Breast Neoplasms↗
PubMed1986

The detection of oligoclonal IgG bands in unconcentrated cerebrospinal fluid by isoelectric focusing on thin-layer agarose gels and silver staining.

Isoelectric focusing on thin-layer agarose gels with silver staining was used to detect oligoclonal bands of IgG in unconcentrated cerebrospinal fluid (CSF). Comparison of this procedure with Coomassie blue staining for the detection of oligoclonal bands in CSF yielded concordant results; CSF samples were either positive or negative for oligoclonal bands by both techniques. The silver-staining procedure was capable of detecting oligoclonal bands in CSF samples with IgG levels as low as 2 mg/dL, which was 200 ng/10-microliter sample. False-positive results were not detected in CSF samples with either a high initial IgG level or a low initial IgG level that required concentration before analysis, even by silver staining. This procedure has several advantages over conventional assays: it is fast, needing only five hours; it uses only 10 to 15 microliter of sample; it is easy to perform; and it can be done routinely in the clinical laboratory.

Gels↗
PubMed1994

Sequential quantification of AgNOR area and number during silver staining by means of an image analysing system.

Standardized AgNOR staining protocols are required to compare results obtained by means of image analysis systems (IAS) in different laboratories. In order to investigate the staining kinetics of the NOR silver staining, we evaluated automatically the area and number of AgNORs of a human transitional-cell carcinoma cell line (HOK-1) by IAS at one minute intervals over a total staining period of 30 minutes. Results showed a constant increase (standard error of estimate: 0.001; R squared: 0.8) of the AgNOR area reaching a plateau after 10 minutes followed by further area increase. In contrast, in all experiments a pronounced variability in the AgNOR number (standard error of estimate: 0.042; R squared: 0.75) was noted, reflecting the aggregation of silver stained spots. Thus the evaluation of the AgNOR area yielded reliable and easily reproducible results, whereas the assessment of the AgNOR number by means of an automated IAS was associated with a marked variability.

Autoanalysis↗
PubMed1993

Improvements in the silver-staining technique for nucleolar organizer regions (AgNOR).

The silver-staining technique for nucleolar organizer regions (AgNOR) of Ploton et al., as popularized by Crocker, is being widely used for evaluation of nucleolar function, especially in neoplasia. It suffers from limited reliability, background staining, precipitates, and fading of the sections. Factors were identified that affect these problems. The oxidation-reduction level and gelatin used are particularly important. An improved procedure is presented which incorporates pre-reduction of the sections, selection of an optimal gelatin, and post-treatment of the sections to produce a permanent preparation. It is compatible with many fixatives and with other stains used before or after the silver stain.

Chemical Precipitation↗
PubMed2001

New temperature modification makes the Bielschowsky silver stain reproducible.

The Bielschowsky silver impregnation can be used as a single stain for the diagnosis of Alzheimer's disease. It impregnates both the amyloid and neuritic components of the senile plaques and it reliably stains neurofibrillary tangles. However, we find that the stain is highly sensitive to changes of the ambient temperature. Since the diagnostic criteria for Alzheimer's disease rely on a semi-quantitative assessment of neuritic plaques and neurofibrillary tangles the reproducibility of the Bielschowsky stain is important. In this study we compare three different modifications of the Bielschowsky stain with the methenamine silver stain and with immunohistochemistry for the beta-amyloid and the hyperphosphorylated tau proteins. We found that the most reliable and easily reproducible modification of the Bielschowsky silver impregnation is the one that uses incubations at 5 degrees C.

Alzheimer Disease↗
PubMed1980

Synaptonemal complex karyotyping in spermatocytes of the Chinese hamster (Cricetulus griseus). IV. Light and electron microscopy of synapsis and nucleolar development by silver staining.

Synaptonemal complexes (SCs), X and Y axes, and various nucleolar structures stain preferentially with silver in surface microspread preparations and are analyzable by both light and electron microscopy. Central elements, kinetochore region material and nuclear annuli which stain with ethanolic phosphotungstic acid are seldom visible after silver staining. SCs can be characterized by length measurements equally well in light and electron micrographs, from which stages of pachytene can also be determined by differentiation of the axes of the XY pair. By electron microscopy, the lateral elements appear as single strands at zygotene and early pachytene, then become double in a plane perpendicular to that of the SC and appear denser and thicker until late pachytene when they become progressively more attenuated and again appear single. These transitions are difficult to explain in terms of separation of associated chromatids. Identification of various silver stained bodies as nucleoli is supported by their orange-red fluorescence with acridine orange. SCs, X and Y axes and associated sex body material are, with a few exceptions, virtually indistinguishable from the background yellow-green fluorescence of the chromatin. Comet-shaped nucleolar bodies are regularly associated with five (in one animal) or six (in two animals) SCs; their positions along particular SCs identifiable by relative lengths indicate these bodies to be expressions of nucleolus organizer regions. They first appear at leptotene in association with unpaired axes and undergo progressive changes through late pachytene, at which time they redistribute their contents coincident with disappearance of the SCs. A characteristic nucleolar double dense body appears at zygotene; unlike the comet-shaped nucleoli, it is unassociated with other nuclear structures, and is assumed to arise from coalescence of previously existing smaller dense bodies. - The silver staining method described is remarkable for the speed and simplicity with which large numbers of spermatocyte nuclei are obtainable for light and electron microscopy. The fidelity of the light microscopic counterpart of the electrom microscopic image has been directly assessed at different stages of pachytene. For cytogenetic analysis, critical information often lies beyond the limits of light optical resolution; the correlated electron microscopy required for verification is easily obtained with this method.

Animals↗
PubMed1997

HpSS: a new silver staining method for Helicobacter pylori.

AIMS: To verify whether the proposed new silver staining method compares favourably with other well established methods in the detection of Helicobacter pylori in gastric biopsies. METHODS: One hundred and forty pairs of antral and fundic biopsies, routinely formalin fixed and paraffin wax embedded, from 70 consecutive unselected patients were stained with haematoxylin and eosin, modified Giemsa, and the proposed H pylori silver stain (HpSS). H pylori immunodetection was performed in the same material with a polyclonal antiserum against H pylori. RESULTS: H pylori was detected in 89 biopsies from 48 patients with haematoxylin and eosin; in a further five biopsies (one antral and four fundic) with Giemsa stain, thereby identifying one more H pylori infected patient. The new silver staining method was positive in all the cases detected by these two methods and detected three extra infected patients (five more positive biopsies). Immunohistochemistry detected one more positive case (two positive biopsies) not identified by any of the other methods. CONCLUSIONS: The HpSS method proposed is highly sensitive in detecting H pylori; it is simple and it compares well with other methods used routinely for evaluating gastric biopsies for H pylori.

Biopsy↗
PubMedDate not supplied

Silver staining of selected mitoses by physical development.

A silver staining method using light-insensitive physical developer for electron microscopic study of selected mitoses is described. The G-banded mitoses are stained with silver by a physical development process that preserves the original G-banding pattern. Transferring the light microscopically selected mitoses to grids provides an examination by optional high resolution in an electron microscope. The physical developer makes the staining standardizable and reproducible, and the intensity of the staining can be controlled by varying the time of development.

Azure Stains↗
PubMed1983

Silver staining of proteins in polyacrylamide gels: increased sensitivity by a blue toning.

An image amplification of silver-stained proteins in polyacrylamide gels by a blue toning has been developed. Following the silver staining, this technique is fast, simple, requires only stable and inexpensive reagents, and its sensitivity is three- to sevenfold higher than the silver staining alone, and 300- to 700-fold higher than the Coomassie blue. When combined with a silver staining utilizing dithiothreitol (J. H. Morrissey, (1981) Anal. Biochem. 117, 307-310) this method is shown to be uniform in sensitivity for five standard proteins. The blue toning does not affect the linearity of the staining and allows the quantitation of proteins by densitometric evaluation.

Animals↗
PubMed1988

Transfer of proteins from acrylamide gels to nitrocellulose paper after silver stain detection.

A method is described for the silver staining of polyacrylamide gels and transfer to nitrocellulose sheets which couples the advantages of silver stain detection sensitivity with efficient transfer to nitrocellulose sheets. Using this procedure, a detection sensitivity of 10 ng was achieved while retaining a transfer efficiency for most proteins that is equivalent or better than methodology that does not permit prior visualization of protein. When coupled with the use of an acrylamide template, this procedure permits protein detection, simultaneous transfer of a large number of cored protein products from many one- or two-dimensional polyacrylamide gels to a single sheet of nitrocellulose, and retention of immunoreactivity. Prior detection, simultaneous transfer, and retention of immunoreactivity provide a substantial number of biochemical and technical advantages.

Collodion↗