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PubMed1981

Further studies on the cytochemistry of the standardized silver staining of interphase nucleoli in smear preparations of Yoshida ascitic sarcoma cells in rats.

Silver staining procedure for the selective demonstration of nucleolar silver stained granules (SSG) and for the simultaneous demonstration of SSG and nucleolar silver stained matrix (SSM) were studied in smears of rat Yoshida sarcoma cells. The successful results of these procedures depend mainly on the quality of silver nitrate and formaldehyde. However, both chemicals can be easily standardized and stabilized disregarding their origin and batch. In standardized procedures (one-step procedure for the selective demonstration of SSG and two-steps procedure for the simultaneous demonstration of SSG and SSM) the silver is apparently bound to acidic groups of proteins of SSG and SSM. The proteins of SSG and SSM seem to be different but both belong to the group of acidic non-histone proteins. According to the results of digestion experiments a possibility also exists that the acidic proteins of SSG may be associated with DNA. The identification of SSG visualized by described standardized procedures was determined not only by cytochemical extraction tests but also by biological experiments. The latter demonstrated that the number of SSG in Yoshida sarcoma cells decreases after treatment of experimental animals with actinomycin D and therefore depends on the state of the nucleolar RNA synthesis.

Animals↗
PubMed2001

Expressions of proliferation markers (Ki-67, proliferating cell nuclear antigen, and silver-staining nucleolar organizer regions) and of p53 tumor protein in gestational trophoblastic disease.

OBJECTIVE: This study was undertaken to determine whether the expressions of 3 proliferation markers (Ki-67, proliferating cell nuclear antigen, and silver-staining nucleolar organizer regions) and of p53 tumor protein could differentiate spontaneous abortions from gestational trophoblastic diseases and also discriminate among gestational trophoblastic disease subgroups. STUDY DESIGN: Twenty-two partial hydatidiform moles, 17 complete hydatidiform moles, 6 invasive hydatidiform moles, and 20 nonhydropic spontaneous abortions (control group) were evaluated by means of immunohistochemical techniques with antibodies to Ki-67, proliferating cell nuclear antigen, and p53. One-step silver staining was used to detect silver-staining nucleolar organizer regions. RESULTS: The expressions of Ki-67, proliferating cell nuclear antigen, silver-staining nucleolar organizer regions, and p53 were significantly higher in the gestational trophoblastic disease group than in the control group. The results of linear discriminant analysis showed that silver-staining nucleolar organizer region count had the highest sensitivity and specificity (93.3% and 100%, respectively) for distinguishing gestational trophoblastic disease from spontaneous abortion. Sensitivity and specificity for discriminating gestational trophoblastic disease from spontaneous abortion increased to 100% when all four markers were used together. Proliferating cell nuclear antigen was found to be the best discriminating variable for differentiating among gestational trophoblastic disease subgroups. CONCLUSION: Our findings suggest that expressions of Ki-67, proliferating cell nuclear antigen, silver-staining nucleolar organizer regions, and p53 may aid in the diagnosis of gestational trophoblastic diseases. These fairly rapid, simple, and economic techniques could serve as a useful adjunct to conventional methods in the diagnosis of gestational trophoblastic diseases.

Abortion, Spontaneous↗
PubMed1990

Quantity of nucleolar silver-stained proteins is related to proliferating activity in cancer cells.

The relationship between the silver-stained proteins of interphase nucleolar organizer regions (NORs) and proliferative activity was studied in 12 established cell lines derived from different tumor types. Quantitative evaluation of NOR proteins was carried out on silver-stained cells, by means of an automated image analyzer and the cell proliferation rate was measured with [3H] thymidine labeling. A linear relationship was found between cell duplication activity and the amount of silver-stained NOR proteins independently of the type of cancer cells (r = 0.94, p less than 0.001). Measurement of silver-stained NOR protein could thus be used as a simple estimate of cancer cell proliferation rate.

Biomarkers, Tumor↗
PubMed1983

Silver staining of histones in Triton-acid-urea gels.

A reliable method for silver staining histones in Triton-acid-urea gels was developed. Optimum staining is achieved by treating the gels either with amido black or a colorless, water-soluble analog of amido black, 2,7-naphthalenedisulfonic acid, prior to staining with ammoniacal silver. Staining of purified calf thymus histones H2A, H2B, H3, and H4 by this method is 30 times more sensitive than staining with amido black alone, allowing the detection of each histone and its modified forms down to the nanogram level. The use of 2,7-naphthalenedisulfonic acid dramatically shortens the procedure permitting histone patterns to be visualized within 5 h.

Acetates↗
PubMed1984

Silver staining, immunofluorescence, and immunoelectron microscopic localization of nucleolar phosphoproteins B23 and C23.

Nucleolar organizer region (NOR)-specific silver staining and immunolocalization of nucleolar phosphoproteins B23 and C23 were compared in Novikoff hepatoma ascites cells. Silver staining and protein C23 immunostaining were both localized in the fibrillar shell surrounding the fibrillar center and in the fibrillar center. During mitosis, silver staining and protein C23 were localized at the NORs. Therefore, protein C23 and the silver-staining protein both seem to be associated with rDNA-containing structures (Mirre and Stahl 1981). A comparison of toluidine blue staining specific for RNA and B23 immunostaining demonstrated that protein B23 was associated with RNA-containing regions of the nucleolus and was absent from the fibrillar centers. Localization of these proteins and their functions are discussed in relation to the organization of the nucleolus.

Animals↗
PubMedDate not supplied

Consecutive silver staining and autoradiography of 35S and 32P-labeled cellular proteins: application for the analysis of signal transducing pathways.

The methodology for the simultaneous analysis of protein synthesis concomitant with protein phosphorylation/dephosphorylation is described. The technique consists of metabolic labeling of rat liver epithelial (RLE) cells with [32P]orthophosphate and [35S]methionine, performing two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) of the mixed samples, followed by silver staining and subsequent autoradiography of the dried silver stained 2-D PAGE electrophoretograms using two films placed back-to-back. The first film, which is positioned in direct contact with the dried silver-stained gel, visualized both exposure to 35S and 32P while the second film recorded exposure to only 32P due to the differential energy levels of the two isotopes. The juxta-positioning of the silver-stained images with the two autoradiographic film images permits the unambiguous mapping of the phosphorylated polypeptides back to their corresponding silver-stained and methionine-labeled counterparts. This strategy provides quantitative information utilizing both silver staining (measure of constitutive levels of protein expression) and metabolic labeling to measure rates of protein synthesis and/or degradation and phosphorylation and/or dephosphorylation using [35S]methionine and [32P]orthophosphate, respectively. We have utilized this methodology for the in vitro analysis of transforming growth factor type beta 1 (TGF-beta 1)-mediated signal transduction in RLE cells and have identified three nuclear polypeptides, 1 (pI 4.95/M(r) 97 kDa), 2 (5.00/85 kDa) and 3 (4.90/84 kDa) whose phosphorylation status is rapidly and transiently modulated by TGF-beta 1. The methodology described should have wide applications in studies where it is desirous to measure protein synthesis and/or degradation concomitant with signal transduction pathways involving protein phosphorylation.

Animals↗
PubMed1988

A nondenaturing vertical isoelectric focusing polyacrylamide slab gel system suitable for silver staining and electrophoretic blotting.

We have devised a nondenaturing vertical isoelectric focusing (IEF)-polyacrylamide gel electrophoresis (PAGE) system which is amenable to silver staining and electroblotting. Apart from being accessible, inexpensive, and simple to use, this new methodology overcomes problems inherent in current IEF methods, for example, pH gradient drift, nonuniform cooling, restricted sample volume, and inability to perform electroblotting. Two photopolymerization gel formulas were derived: a 5% acrylamide formula using bisacrylamide (Bis) as the crosslinker and a 6% acrylamide formula using diallyltartdiamide (DATD) as the crosslinker. The 5% acrylamide Bis gel gave excellent resolution and separation of proteins whereas the 6% acrylamide DATD gel expanded slightly during silver staining, resulting in mild band distortions. At least 80 ng of protein per band could be detected by the silver staining protocol devised. Both the DATD and the Bis gels were suitable for electroblot transfer. Parameters to ensure the optimum conditions for reproducible, high resolution vertical IEF-PAGE are described. IEF-PAGE silver staining and electroblotting procedures and silver staining of the nitrocellulose electroblot procedures are also described. The advantages of this methodology over previously published methods are discussed.

Animals↗
PubMed2000

A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry.

The growing availability of genomic sequence information, together with improvements in analytical methodology, have enabled high throughput, high sensitivity protein identification. Silver staining remains the most sensitive method for visualization of proteins separated by two-dimensional gel electrophoresis (2-D PAGE). Several silver staining protocols have been developed which offer improved compatibility with subsequent mass spectrometric analysis. We describe a modified silver staining method that is available as a commercial kit (Silver Stain PlusOne; Amersham Pharmacia Biotech, Amersham, UK). The 2-D patterns abtained with this modified protocol are comparable to those from other silver staining methods. Omitting the sensitizing reagent allows higher loading without saturation, which facilitates protein identification and quantitation. We show that tryptic digests of proteins visualized by the modified stain afford excellent mass spectra by both matrix-assisted laser desorption/ionization and tandem electrospray ionization. We conclude that the modified silver staining protocol is highly compatible with subsequent mass spectrometric analysis.

Animals↗
PubMed1988

'Catalysts' for polyacrylamide gel polymerization and detection of proteins by silver staining.

The crosslinker diacrylyl-piperazine produces polyacrylamide gels which display improved electrophoretic separation of proteins and better physical strength. It also produces gels with improved detection of proteins by ammoniacal silver staining by reducing the background. This reduced background provided us with an opportunity to investigate residual background staining caused by the catalytic reagents utilized in the polymerization of acrylamide gels. The commonly used catalyst system, tetramethyl-ethylenediamine and ammonium persulfate was shown to be responsible for the yellow staining background found after a prolonged development time with silver staining. An alternate catalyst system has been designed to decrease further the formation of this background staining. Dimethyl-piperazine or tetramethylethylenediamine, potassium or ammonium persulfate, and sodium thiosulfate are shown to provide for gels which have excellent mechanical and staining characteristics. These catalytic systems produce little background staining despite prolonged development time with the ammoniacal silver stain, and they reduce background staining with the dichromate silver stain.

Acrylic Resins↗
PubMed1997

Age-related changes of sulphide-silver staining in the rat hippocampus.

The influence of ageing on sulphide-silver positive zinc stores was assessed in the stratum radiatum of the CA1-CA3 sub fields of the rat hippocampus and in the molecular layer of the dentate gyrus using a silver amplification histochemical technique associated with microdensitometry. The volume of areas examined for microdensitometry was evaluated as well by quantitative image analysis. Male Sprague-Dawley rats aged 3 months (considered to be young), 12 months (considered to be adult) and 24 months (considered to be old) were used. Microdensitometric analysis of values of sulphide-silver staining corrected for the volume of hippocampal areas investigated revealed no age-dependent changes of staining in the CA1 sub field of the hippocampus. In the CA2 sub field a decrease of sulphide-silver staining was noticeable in aged rats in comparison with younger cohorts. A progressive reduction in the intensity of sulphide-silver staining was observed in the CA3 sub field of the hippocampus. In the molecular layer of the dentate gyrus, the intensity of staining was decreased in adult and old rats in comparison with young animals. These findings indicate a different sensitivity to ageing of histochemically detectable zinc stores of rat hippocampus. The possibility of a specific sensitivity to senescence of different zinc-containing pathways of the hippocampus is discussed.

Aging↗
PubMed1983

Silver staining for nucleolar organizing regions of vertebrate chromosomes.

Refinements to a simple, one-step silver staining technique for nucleolar organizing regions are described. These include fixation of silver stained material with sodium thiosulfate and standardization of silver development conditions for different groups of vertebrates. The central advantages to the method are that it is rapid, reliable, simple, and inexpensive. Additional benefits include (i) consistent and uniform silver staining of nucleolar organizing regions, (ii) few reduced silver deposits elsewhere on the chromosomes or on the slides, (iii) generally unaltered chromosome morphology after silver treatment, and (iv) relative permanence of Permounted preparations. The method works equally well on chromosomes made from cell cultures and from solid tissues of live specimens.

Animals↗
PubMed1998

[DNA sequencing of human ciliary neurotropic factor gene by silver staining method].

OBJECTIVE: To introduce a non-radioactive protocol for DNA sequence analysis which employs a silver staining procedure METHODS: DNA template is amplified by Taq DNA polymerase to generate sequence ladder, No labeled nucleotide or primer is involved in the thermal-cycling sequencing reaction. A sensitive silver staining procedure is employed to visualize bands in sequencing gel. RESULTS: This silver staining method is used for DNA sequence analysis of human cilliary neurotrophic factor (hCNTF) gene. The resulted band resolution is comparable to radioactive sequencing method, and data can be obtain within less than 2 hours after sequencing reaction. CONCLUSION: Silver staining is a rapid, inexpensive and reliable method for DNA sequence analysis.

Ciliary Neurotrophic Factor↗
PubMed1992

Intensification of labelings of the immunogold silver staining method by gold toning.

We evaluated the applicability of the gold toning procedure to the immunogold silver staining method using monoclonal antibody against dopamine. Immunolabeling was examined in the rat substantia nigra at light and electron microscopic levels. Vibratome sections of fixed midbrains were incubated with anti-dopamine antiserum and then with 5 nm colloidal gold bound to goat anti-mouse immunoglobulin. Silver staining of these sections produced a light brown immunolabeling. After the sections were processed by gold toning, the labeling became intense black. At a light microscopic level, these high contrast signals were retained after the sections were osmicated and embedded in Epon. At an electron microscopic level, signal-to-noise ratio was high, and the positive staining could easily be verified at a low-power magnification. The technique described here, the gold-toned immunogold silver staining method, provides high contrast signals and is much more sensitive than immunogold silver staining alone. This method, therefore, has great potential for use in immunohistochemical analysis of the central nervous system.

Animals↗
PubMed1986

Detection of antigen in immune precipitates by silver staining of SDS-polyacrylamide gels.

The silver staining technique of Merril et al. (1981) was used to identify antigens separated by SDS-PAGE after dissociation of immune complexes purified by absorption with staphylococcus A protein. The five and four dominating structural components of measles and RS virus (Mr range 28,000 to 79,000) respectively precipitated with monoclonal antibodies were readily identified, except when the Mr of the virus-specific antigen was in the same range as immunoglobulin heavy and light chains. Specific antigens were identified less effectively after precipitation with polyclonal sera because of a larger background of stainable proteins. This method, which combines the sensitivity and high resolution of silver staining and the specificity of immune complexing with monoclonal antibodies, is rapid and inexpensive.

Antibodies, Monoclonal↗
PubMed1988

Coloration of silver-stained protein bands in polyacrylamide gels is caused by light scattering from silver grains of characteristic sizes.

This study investigates the physical basis of color effects in the detection of proteins in polyacrylamide gels by silver staining. Specifically, the hypothesis that different colors may correlate with the development of silver grains of characteristic sizes was investigated by electron microscopy. Protein bands that stained brown, yellow, and blue were excised from stained gels and prepared for electron microscopy by thin-sectioning. In each case, the size distributions of globular silver grains were determined directly from the electron micrographs. We found that blue bands have larger silver grains (with diameters of 40-100 nm) than yellow (21-39 nm) or brown bands (17-35 nm). On the basis of these and other observations, a general mechanism is proposed whereby chemical specificity of electrophoretically separated proteins is expressed in color-specific silver staining.

Electrophoresis, Polyacrylamide Gel↗
PubMed1991

Detecting proteins containing 3,4-dihydroxyphenylalanine by silver staining of polyacrylamide gels.

Proteins in which some or all of the tyrosine side chains are post-translationally modified to dihydroxyphenylalanine have been found in several invertebrate phyla. In this paper we describe the unusual silver-staining properties of these 3,4-dihydroxyphenylalanine (Dopa)-proteins in silver-stained polyacrylamide gels. Our evidence suggests that the rapid silver staining of these proteins is due to the 3,4-dihydroxyphenol ring which is a highly effective reducing agent in the alkaline development conditions used in the final step of most silver-staining procedures. Normal proteins comprising the standard 20 amino acids and tyrosine on its own, do not reduce silver under these conditions. Pretreatment of the gels with acid-dichromate solutions abrogates the rapid staining of the Dopa-proteins. This rapid silver-staining technique will facilitate the rapid screening of many additional organisms for Dopa-proteins using sodium dodecyl sulfate gels and small amounts of tissue.

Animals↗
PubMed1983

Ammoniacal silver staining of proteins: mechanism of glutaraldehyde enhancement.

When the conditions for detecting proteins by ammoniacal silver staining (B. R. Oakley, D. R. Kirsch, and N. R. Morris (1980) Anal. Biochem. 105, 361-363.) following gel electrophoresis were varied, it was noted that glutaraldehyde pretreatment was necessary for maximal staining, which could not be explained simply as the result of "fixation." Further studies indicated that glutaraldehyde enhancement of protein staining with this silver reagent was probably due to oxidation of the aldehyde groups by silver ions, resulting in metallic silver depositions within the gel which act as nucleation sites for additional metallic silver localization in the protein bands upon the addition of formaldehyde developer. This proposed mechanism is consistent with the Tollen's reaction, as well as some aspects of the photographic process. Consistent with this notion, silver-staining intensities are directly related to mole percentage lysine of various standard proteins.

Aldehydes↗
PubMed1981

Nucleolar silver stained granules in rat Yoshida sarcoma cells after RNA synthesis inhibition.

The number of nucleolar silver stained granules representing nucleolus organizer regions in interphase nuclei was studied in Yoshida ascitic sarcoma cells without and after the inhibition of the nucleolar RNA synthesis with actinomycin D or cyclophosphamide. The results demonstrated that the number of nucleolar silver stained granules decreased after the inhibition of the nucleolar RNA synthesis with these drugs disregarding the mode of their action. In addition, the decrease of nucleolar silver stained granules in number produced by actinomycin D was dose dependent. Similarly, the number of nucleoli without silver stained granules increased depending on the dose of the administered actinomycin D.

Animals↗