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PubMed1986

Requirement for cysteine in the color silver staining of proteins in polyacrylamide gels.

To determine whether cysteine residues have a contribution to the mechanism of color silver staining, we silver stained sodium dodecylsulfate polyacrylamide gel electrophoresis separations of proteins which have few or no cysteines. Proteins without cysteine stained negatively (yellow against a yellow background) with silver. Proteins with one or more cysteines stained orange, red, brown, or green/gray depending on the mole percentage of cysteine and whether they contained covalently attached lipids. The colors could not be correlated with the mole percentages of cysteine of these proteins indicating that some components other than cysteine affect the staining color of cysteine-containing proteins. Silver staining of amino acids, sugars, nucleotide bases, or lipopolysaccharide dot-blotted onto nitrocellulose paper implicated adenine, lipids, the basic amino acids, and glutamine, but not sugars or other amino acids in silver/protein complexes.

Bacterial Outer Membrane Proteins↗
PubMed1981

Familial silver staining patterns of human nucleolus organizer regions (NORs).

The silver staining patterns of the nucleolus organizer regions (NORs), an indication of rDNA transcriptional activity, were studied in metaphases from lymphocyte cultures of 20 karyotypically normal members of three families selected for a large sibling number or a monozygotic twin pair. Quinacrine polymorphic markers and bands were used to identify the acrocentrics and to determine their parental origin. A comparison of the silver staining frequencies among siblings and between parent and child indicated no significant differences for any acrocentric in the twin pairs and significant differences (P less than .05) for only one of the 20 acrocentrics segregating in each of two families. These two acrocentrics had short stalks with very small silver deposits (AgNORs). The mean size of the AgNOR, based on a relative score, was not significantly different (P greater than .05) for each homolog between the twin pair and in approximately 70% of the acrocentrics shared by members of the one family analyzed. The frequency with which a particular chromosome was silver stained demonstrated a significant correlation (r2 = .732) with the size of AgNOR. There was a close correlation (r2 = .609) between stalk length and the size of the AgNOR. We conclude that the frequency of silver staining and the mean size of the AgNOR are characteristics inherent in a particular chromosome carried from one generation to the next.

Cells, Cultured↗
PubMed1983

Silver staining of histone-depleted metaphase chromosomes.

To investigate a possible relationship between the core-like structures seen in silver-stained chromosomes (prepared by standard cytogenetic methods) and the scaffolds observed in histone-depleted chromosomes, the ability of the scaffold to stain with silver has been examined. Isolated chromosomes were histone-depleted by washing in ammonium acetate or by spreading the chromosomes on an ammonium acetate hypophase. The residual chromosome structures were carbon-platinum shadowed or stained with silver, and then examined by electron microscopy. The results provide clear evidence that the scaffold structure has a high affinity for silver and is therefore similar in its silver-staining potential to the core structure in standard chromosomes. This suggests that the silver core in standard chromosomes may represent the scaffold visualized by histone depletion. The peripherally dispersed DNA radiating from the scaffold also proved to be silver-reactive, and additional experiments demonstrated that purified DNA is capable of binding silver. This result indicates that cytological silver staining is not simply a matter of staining protein, as has previously been thought, but may also involve the staining of chromosomal DNA. In the ammonium acetate-treated and carbon-platinum-shadowed preparations, the scaffold structure was highly variable in its morphology and appeared to be composed of undispersed or incompletely dehistonized chromatin fibers. The silver-stained scaffold reflected this variability. Taken together with other evidence, these findings lead to a questioning of the reality of chromosome core structures.

Acetates↗
PubMed1982

Electron microscopy of silver-stained core-like structures in metaphase chromosomes.

Chinese hamster metaphase chromosomes, stained with ammoniacal silver and examined by electron microscopy, were covered with fine silver grains of variable size. In addition, many chromosomes contained linear aggregates of silver grains running continuously from one end of each chromatid to the other, forming a core-like structure. Extensive deposits of silver were observed over the nucleolar organizers and the centromeric regions, and the silver precipitate in the latter region appeared to be a localized differentiation of the core-like structure. The silver cores ranged from thin to thick and continuous to discontinuous elements in different chromosomes. This extreme variability suggests that these cores are not true structural components of chromosomes. The amount of silver deposit over any given chromosome region may simply reflect the concentration of chromatin in that region of the chromosome. The silver-stained core-like structure probably reflects an underlying difference in the concentration of chromatin in the central and peripheral region of each chromatid. Such differences in chromatin concentration may be induced during the prolonged hypotonic treatment required for the subsequent visualization of cores.

Animals↗
PubMedDate not supplied

Comparison of immunohistochemistry and silver stain for the diagnosis of pediatric Helicobacter pylori infection in urease-negative gastric biopsies.

We compared immunohistochemical and silver stains of pediatric gastric biopsy sections for the identification of Helicobacter pylori infection with chronic inflammation and a negative urease screening test. Thirty-seven patients (age range 10 months to 21 years) whose gastric antral biopsies were negative for the rapid urease test (CLO(R)) but positive for lymphocytic infiltration were selected for a retrospective study. Specimens had been subjected to a rapid urease test (CLO(R)) and hematoxylin and eosin staining, and Dieterle silver staining and immunohistochemical staining specific for H. pylori were also performed. Twelve additional patients with urease-positive biopsies were used as controls. With Dieterle staining, 8/37 (22%) urease-negative biopsies contained organisms morphologically compatible with H. pylori, 21/37 (56%) contained organisms not compatible with H. pylori, and 8/37 (22%) were negative for organisms. Immunostaining confirmed 6/8 (75%) Dieterle-positive cases as being H. pylori, was negative in 2/8 (25%) Dieterle-positive cases, and was positive in 2/8 (25%) Dieterle-negative cases. Biopsies from 8/12 (67%) urease-positive specimens contained organisms seen with both Dieterle and immunohistochemical stains, and 4/12 (33%) were negative with both stains. Although both stains yielded comparable results with H. pylori-positive biopsies, Dieterle staining was potentially confusing because of nonspecific staining of other organisms. A significant proportion of (CLO(R))-negative biopsies was positive for H. pylori with special stains. We therefore recommend the use of immunohistochemical staining rather than silver staining in the evaluation of urease-negative gastric biopsies demonstrating chronic inflammation in children.

Adolescent↗
PubMed1985

Ultrastructural localization of nucleolar material by a simple silver staining technique devised for plant cells.

A simple silver staining technique for use at the electron microscopic level, consisting only of treatment with aqueous silver nitrate at high temperature for a prolonged time, was applied to thin sections of root tip meristems of Vicia faba. This technique contrasted the fibrillar component and the granular component in interphase nucleoli as a reflection of the degree of packing. In contrast, silver impregnation was scarcely discerned in chromosomes. A comparison of silver staining and conventional double staining showed that the fibrillar centres did not always respond positively to silver. During the course from metaphase to late anaphase the nucleolus organizing secondary constriction was always seen as a heavily impregnated region and the electron density of the cytoplasm increased, probably due to dispersed nucleolar material. An argyrophilic substance began to accumulate on chromosomes in late anaphase. In the beginning of telophase a uniformly impregnated nucleolus was formed at the secondary constriction. It is concluded from these results that argyrophilic substance is associated with RNA-containing structures rather than DNA-containing structures. The silver staining technique presented here is very convenient and favourable, especially for plant cells, to detect specifically the nucleolus organizing region and to survey the nucleolar material during mitosis at the electron microscopic level.

Cell Nucleolus↗
PubMed1985

Stereoscopic back-scattered electron imaging of silver-stained proteins in nucleoli.

By using simultaneously the AgNOR silver staining method, back-scattered electron imaging mode and stereo-tilt in scanning electron microscopy (SEM), it is possible to observe the nucleus through the cell surface, the nucleolus, and the tri-dimensional distribution of the Ag-NOR-associated acidic proteins. In C3H10T1:2 cells and their 7-12-dimethylbenz-alpha-anthracene-treated transformants, the staining demonstrates several intranucleolar silver-staining granules (SSG), surrounded by a weakly staining region. The SSG may represent the fibrillar center (FC) and the weakly staining region, the fibrillar dense component (FD). This component can link several SSG together to form a "rope-like structure". In cells with no visible nucleolus and inactive nucleolar organizer regions (NORs) the silver-staining granules are less numerous, close together and the presumed fibrillar dense components are not visible. The SSG are located more peripherally, and the weakly staining region and the "rope-like structure" are less prominent in control cell nucleoli than in transformed cells with a comparatively high rate of RNA synthesis.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1988

Agarose gel electrophoresis of denatured RNA with silver staining.

This paper describes agarose gel electrophoresis and silver staining of denatured RNAs. Glyoxal- or formaldehyde-denatured RNAs are electrophoresed in an agarose gel cast on a plastic support using an inert, low conductivity buffer. Following electrophoresis, the gel is stained with a sensitive silver stain. The method produces sharp, well-resolved bands and yields accurate RNA size estimates. Because of its sensitivity and simplicity, it is suitable for routine laboratory use.

Electrophoresis, Agar Gel↗
PubMed2004

Sensitive silver staining of protein in sodium dodecyl sulfate-polyacrylamide gels using an azo dye, calconcarboxylic acid, as a silver-ion sensitizer.

A highly sensitive silver staining method for detecting proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was developed. It is based on the silver nitrate staining method but also employs an azo dye, calconcarboxylic acid (NN), as a silver-ion sensitizer. It increases silver binding on protein bands or spots by the formation of a silver-dye complex and also increases the reducing power of silver ions to metallic silver by NN itself with formaldehyde. After a 2 h gel fixing step, the protocol including sensitization, silver-ion impregnation, and reduction steps can be completed in 1 h. The sensitivity is superior to that of silver stain with glutardialdehyde as a silver-ion sensitizer. The detection limit of NN-silver stain is 0.05-0.2 ng protein. Considering the high sensitivity without using glutardialdehyde, the NN-silver stain would be useful for routine silver staining of proteins.

Azo Compounds↗
PubMed2005

Effect of glass dissolution products on the detection of proteins by silver staining.

The influence of glass dissolution on the silver staining of proteins was investigated by reacting glass microspheres of varying chemical durability in boiling Laemmli sample buffer (LSB) for up to 5 min. All three of the investigated glass compositions leached Na+ ions to varying degrees during boiling in LSB, thereby causing an increase in the pH of the sample buffer. The LSB supernatant from the dissolution tests was mixed with unreacted LSB containing human serum albumin (HSA) and standard one-dimensional SDS-PAGE was performed. Silver staining was then used to visualize protein bands within the gel. The 30 Na2O.70 SiO2 glass exhibited pronounced degradation as shown by scanning electron microscopy. Further experiments employing solutions of neat LSB and reacted LSB (i.e., LSB containing glass dissolution products) mixed at varying ratios demonstrated the apparent significance of sample pH in affecting the inhibition of silver staining. The cause of this behavior may be due to an interference with the fixation stage of the staining protocol, thereby resulting in the loss of protein in subsequent rinsing stages.

Buffers↗
PubMed1982

Demonstration of kinetochores and centrioles in spermatocytes of two species of cockroaches by silver staining.

Light microscopy following silver staining of spermatocytes of German and Madagascar hissing cockroaches demonstrated: (1) the localization of a kinetochore in each autosomal synaptonemal complex during pachytene, and (2) visualization of centrioles in different stages of meiotic prophase. The presence of a "hairpin-like" twist and the nucleolus organizer region in the X-chromosome was observed only in the German cockroach.

Animals↗
PubMed1979

Cytological and histochemical studies on the mechanism of the selective silver staining of nucleolus organizer regions (NORs).

A new silver staining method is presented (Ag-II staining) providing a rapid and reproducible way to selective silver staining of nucleolus organizer regions (NORs). In comparison with other techniques, such as the Ag-AS method and the Ag-I method, factors influencing silver stainability are discussed. Histochemical studies on the nature of the NOR-specific silver precipitate were performed either by employing various pretreatments or by inhibiting the participation ("blocking") of the various proteins or protein compounds in the staining reaction. The results would seem to indicate that the interactions of silver-ions with the carboxyl groups of acidic proteins which are involved in the rRNA-transcription process are mainly responsible for the selective silver staining of NORs.

Animals↗
PubMed1984

Detection of protein in polyacrylamide gels using an improved silver stain.

A much improved silver staining procedure for the detection of protein in polyacrylamide gels of 0.8-3.0 mm thickness is described. It achieves very high sensitivity (detecting less than 0.01 ng bovine serum albumin/mm2) by overstaining and subsequently removing nonspecific background stain using a modified, reliable destaining procedure. Maximum sensitivity follows prediamine equilibration in 0.1% (w/v) formaldehyde solution. With two-dimensional electrophoresis the improved staining procedure reveals greater than 200 polypeptides in unconcentrated human urine and greater than 150 polypeptides in a single human fingerprint.

Electrophoresis, Polyacrylamide Gel↗
PubMed1995

Improved method for silver staining of glycoproteins in thin sodium dodecyl sulfate polyacrylamide gels.

A method for detection of glycoproteins in thin sodium dodecyl sulfate polyacrylamide gels was developed by a combination of (i) initial periodic acid oxidation/Alcian blue staining and (ii) subsequent staining with silver nitrate. The procedure allowed detection of as little as 1.6 ng of alpha 1-acid glycoprotein and 8-40 ng of a polydisperse mucin sample, which is at least 10 times more sensitive than previously published methods. The method should be very useful for assessment of sample purity and detection of glycoproteins in dilute mixed samples.

Alcian Blue↗
PubMed1988

Localization of cutaneous antigens by the immunogold-silver staining technique.

Immunogold-silver staining (IGSS) was evaluated for the localization of cutaneous antigens in frozen and paraffin-embedded tissue, employing antibodies to a variety of intracellular, cell-surface and extracellular epitopes in an indirect immunogold reaction, followed by silver enhancement. The principal advantages of IGSS are the avoidance of toxic reagents and the production of a silver precipitate that is permanent, clearly visible and of sufficient contrast with the reaction products of alternative immunolocalizing techniques to be of value in double-labelling procedures. It seems to localize antigenic determinants well, but does not appear to be particularly sensitive, especially for the demonstration of antibodies to extracellular constituents. Limitations of IGSS include the propensity to non-specific staining, and the need to vary the duration of the development stage during silver enhancement.

Antigens↗
PubMed1987

Tryptic peptide analysis of nanogram quantities of proteins: radioiodination of proteins detected by silver staining in polyacrylamide gels.

The silver-staining procedure for detecting proteins in polyacrylamide gels has been modified so that the polypeptides remain suitable for subsequent radioiodination and tryptic peptide analysis. The procedure, which involves a silver-staining/destaining protocol that minimizes crosslinking, is more rapid than many other methods, and can detect as little as 1 ng of protein. Following elimination of silver, the proteins can be radioiodinated and digested with trypsin by a modification of the method described by J. H. Elder, R. A. Pickett, J. Hampton, and R. A. Lerner (1977, J. Biol. Chem. 252, 6510-6515). Together, these improvements have increased the sensitivity of the tryptic peptide mapping technique by three orders of magnitude and thereby enabled us to perform reproducible structural analysis of femtomolar quantities of proteins.

Electrophoresis, Polyacrylamide Gel↗
PubMed1990

Silver staining of proteins and DNA.

Silver stains offer high sensitivity for the detection of proteins and DNA separated on gels and membranes. These stains depend on the reduction of ionic to metallic silver.

Ammonia↗