Gel protein stains: silver stain.
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Electrophoresis↗
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Some introductory comments on silver staining.
Silver staining has become a versatile method for the visualization of specific cell structures and products. The similarity of the impregnation "nuclei" of reduced silver staining to the silver "specks" or "nuclei" of the latent image in photography is noted. "Physical" development (reduction of ionic silver in solution) in silver staining as compared to "chemical" development (reduction of ionic silver remaining in a silver halide crystal) in photographic procedures is briefly discussed.
Animals↗
Comparative analysis of an improved thioflavin-s stain, Gallyas silver stain, and immunohistochemistry for neurofibrillary tangle demonstration on the same sections.
An improved thioflavin-S stain, Gallyas silver stain, and two immunostainings were quantitatively compared for demonstration of neurofibrillary tangles (NFTs) on the same sections. Sections of hippocampal formation from seven cases of Alzheimer's disease (AD) were immunofluorescently stained with a commercially available polyclonal NFT antibody or a PHF-1 monoclonal antibody, followed by an improved thioflavin-S stain, and finally by Gallyas silver staining. The thioflavin-S method was improved by using a combination quenching method that removes background autofluorescence without remarkable tissue damage and by post-treatment with concentrated phosphate buffer, which minimizes photobleaching. PHF-1 or NFT immunostaining is much less sensitive than the improved thioflavin-S staining and Gallyas silver staining, particularly in the transentorhinal region. Moreover PHF-1 immunoreactivity varied greatly among AD individuals. Thioflavin-S staining and Gallyas silver staining show almost the same sensitivity in NFT demonstration, but only the former depends on the secondary protein structure of NFTs. This study suggests that the improved thioflavin-S staining is a simple, sensitive, and consistent method for demonstration of neurofibrillary pathology.
Aged↗
Determination of poly(ADP-ribose) chain length distribution on polyacrylamide gels by silver staining.
Silver staining and polyacrylamide gel electrophoresis were used to visualize chain length distribution of poly(ADP-ribose) enzymatically synthesized from NAD by rat liver nuclei. The method described has the advantage that synthesis does not require radioactive-labeled NAD, and microgram quantities (greater than 5 micrograms) of poly(ADP-ribose) can be resolved and visualized as discrete bands according to chain lengths which range from 8 to 60 residues. This method can be applied to estimate size distribution of poly(ADP-ribose) chains in cells or tissues.
Chromatography↗
A Drosophila melanogaster cell line tested for the presence of active NORs by silver staining.
Silver staining was used to detect active NORs in a Drosophila melanogaster cell line (C1 82) characterized by dimorphic X chromosomes (XXL), one of the two Xs showing a marked increase in heterochromatin where the nucleolar organizer (NO) is located. The Q-banding technique was used to determine the karyotype characteristics of the line. Ag-positive NORs appeared only on structurally changed X chromosomes (XL), both in diploid and tetraploid cells, indicating that rRNA genes of XL are more active or numerous than those on normal homologues. A possible relationship between NOR stainability, the presence of an increased heterochromatic portion and the selective advantage of XXL cells, recurrent in numerous Drosophila female lines, is discussed.
Animals↗
Analysis of differences between coomassie blue stain and silver stain procedures in polyacrylamide gels: conditions for the detection of calmodulin and troponin C.
It is reported that the conditions used in some silver stain procedures can fail to detect calmodulin, troponin C, and other proteins with similar physical properties. Conditions are described that allow the reproducible detection of these proteins. Two phenomena are described: (1) lack of protein staining when treatment with glutaraldehyde is omitted from the protocol, and (2) loss of small proteins from the gel matrix during prolonged washing procedures. These data directly demonstrate that the use of some silver staining protocols can result in misleading data in biological studies and provide an explanation for at least one class of proteins of how silver staining and Coomassie blue staining of gels can give different results.
Calcium-Binding Proteins↗
The relationship between aging and ribosomal gene activity in humans as evidenced by silver staining.
Silver selectively stains nucleolar organizer regions (NORs) distributed on the D and G chromosomes of man. The number of NORs is fairly constant for a given individual but is highly variable within populations. When 2540 metaphases from lymphocytes were examined from 127 normal subjects, a mean NOR number of 7.3 was obtained with a mode of 7. No significant difference was found in mean NOR number between females and males. However, regression lines do show a decrease in NOR number with aging for both sexes but the rate of decline is more evident in females. Also, G chromosome NORs appear more stable than those in D chromosomes. Since silver--NOR staining is indicative of ribosomal gene activity, it is proposed that lymphocyte rDNA becomes less sensitive to phytohemagglutinin stimulation during aging. It is unique that this gene repression can be visualized with conventional staining and light microscopy.
Adolescent↗
Silver stain cytology; use of silver stain in the detection of uterine cancer by the cytologic method; a survey of over 6000 cases.
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Coloring Agents↗
Recovery of intact proteins from silver stained gels.
Silver stained proteins of a wide molecular weight (MW) range (20-116 kDa) were successfully recovered by both electroblot and electroelution. The recovery was demonstrated for nanogram loads of proteins separated by SDS-PAGE and visualized by silver staining methods compatible and incompatible with mass spectrometry (MS). It was shown that the alcohol/acid and glutaraldehyde fixation steps present in a number of staining procedures did not prevent recovery of intact proteins from gels. It was found that the recovery of intact proteins from silver stained gels was substantially increased upon pre-equilibration in a buffer containing the reducing agent, dithiothreitol (DTT). The effect of destaining on the recovery of silver stained proteins was also investigated. Comparable recovery of intact proteins within a wide MW range from silver stained gels with and without destaining step was demonstrated. Recovery of model proteins from gels visualized using silver staining method compatible with MS showed 52 to 76% yield of that from the unstained gel, depending upon method of the transfer. Comparison of the recovery of intact proteins from gels visualized using other staining procedures was also made. The above findings have implications as to the supposed irreversible nature of protein "fixation" inside polyacrylamide matrix, and confirm lack of binding of proteins in the gel to metal silver deposited on its surface. This method has the potential to be suitable for direct characterization of proteins by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) without additional purification steps.
Electrophoresis, Polyacrylamide Gel↗
Silver staining of proteins on electroblotting membranes and intensification of silver staining of proteins separated by polyacrylamide gel electrophoresis.
A fast and convenient method for silver staining of proteins on electroblotting membranes was developed based on Gallyas' histochemical intensifier and applied to human endothelial cell proteins separated by one- and two-dimensional electrophoresis and electroblotted to polyvinyl difluoride membranes. The method allowed detection of proteins on membranes with a sensitivity equal to the sensitivity of the most sensitive silver-staining protocols for electrophoresis gels. Also, the method was compatible with preceding immunostaining on the same membrane. Furthermore, an intensifying method for proteins in silver-stained SDS-PAGE gels was developed based on Gallyas' histochemical intensifier. This method was applied to proteins separated by one- and two-dimensional gel electrophoresis and visualized by one of several silver-staining methods. Maximal intensification was achieved for the less sensitive but fast acidic silver-staining protocols, but even for the very sensitive alkaline protocols a significant increase in signal to noise ratio was obtained. In particular, negatively stained or invisible proteins on the silver-stained gels were found to be visualized by the Gallyas stain. Proteins from silver-stained and Gallyas-stained gels were identified by mass spectrometry, and the intensification procedure was fully compatible with mass spectrometry.
Cell Line↗
Improved detection of lymphocyte membrane proteins in purified form and as a crude mixture using native and denaturing polyacrylamide gel electrophoresis by optimisation of coomassie brilliant blue and silver staining.
Optimised silver staining protocols were devised for the detection of membrane proteins in purified form and as a crude mixture. These were adduced in both sodium dodecyl sulphate (SDS) and native polyacrylamide gel electrophoresis and consisted of ethanol-acetic acid-formaldehyde fixation, Coomassie Brilliant Blue prestaining, Rapidfix pretreatment, formaldehyde enhancement and finally ammoniacal silver staining. With these modifications, numerous staining problems of membrane proteins were overcome. These included reduction in background staining, enhanced detection sensitivity in native gels, elimination of negative staining and the avoidance of metallic silver deposition on the gel surface. In overcoming these problems, some factors determining the colour and stainability of membrane proteins in their native state were determined. Both the anionic Coomassie Brilliant Blue dye and SDS detergent improved the sensitivity of silver staining in native gels, and ammoniacal silver was more sensitive than neutral silver, suggesting silver staining to be a charge dependent process.
Ammonia↗
The mechanism accounted for the silver staining "fast technique" development and its histochemical meaning: I. The correlation between the silver staining intensity and the tissue bound ferric ion and some other tissue histochemical reactivities.
The mechanism accounted to accomplish the silver staining "fast technique" on tissues sections was studied towards the correlation among histophotometric measures concerning the silver staining intensity and the intensity provided by some histochemical reactions performed on spleen and liver sections from rats and pigs. By treating previously these histological sections with thioglycolate or oxalate solutions in progressive concentrations and afterwards subjecting them to a silver staining "fast technique", it was demonstrated that the silver staining intensity decreases proportionally to the thioglycolate or the oxalate solution concentration. The regression line of the silver staining intensity on the thioglycolate or the oxalate solution concentration was established, as well as its regression coefficient. On the other hand, on histological sections previously subjected to the thioglycolate or the oxalate treatment, some histochemical techniques were performed and the histophotometric measures concerning the intensity of each histochemical technique used were taken and its regression line, as well as its regression coefficient were established. By comparing the silver staining intensity regression line (or its regression coefficient) with the regression lines established for the histochemical techniques used, it was tested the correlation between the silver staining and the reactivity of some reactive groups contained into the tissues. In this manner, the influence of such reactive groups on the silver staining development was tested. The results show that there is no correlation between the silver staining intensity and the reactivity degree of the reducing groups (-SH- and carbonyl group), the 1-2-glycolic group, as well as of some protein reactive groups (phenol, imidazole, carboxylic groups). On the other hand, taking into account the respective regression lines and its regression coefficient, the comparison between the silver staining intensity and the Prussian blue reaction intensity shows a close relationship between them. This finding suggests that for the silver staining "fast technique" the tissue bound ferric ion accomplishes a very important role and can be accounted for its mechanism and its histochemical meaning.
Animals↗
[Humoral protein analysis using a combination of agarose isoelectric focusing and silver staining--experimental evaluation of the silver staining method].
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Body Fluids↗
The mechanism accounted for the silver staining "fast techniques" development and its histochemical meaning. II. The binding between silver and ferric ion accounted for the silver staining development.
Results obtained on filter paper strips models show that some protein and fatty acids become argentophil as an effect of a previously binding ferric ion although, in this instance, the silver staining can only be accomplished by using a silver diamine solution, since a silver nitrate solution is not effective. However, if the filter paper model is previously treated by a "multidentate ligand" before the silver nitrate solution treatment becomes able in doing the silver staining. This result shows that the binding between Fe3+ and Ag+ can be done if a suitable negatively charged "multidentate ligand" has been connected between them. The following "multidentate ligands" were tested: thiocarbohydrazide, carbohydrazide, and hexamethylenetetramine: ammonia was also tested, as an attempt to disclose if it is able to act as a "multidentate ligand". The semicarbazide effect was analysed and compared with the "ligands". It was found, either on filter paper strip models or on histological sections, that every "multidentate ligand", as well as ammonia, are effective in producing silver staining whereas semicarbazide is devoid of effect. The "ligands" effectiveness depends upon the solution pH, in the same manner that is does occur when the silver diamine "fast technique" is used on tissue sections. Histophotometric measures, taken on tissue sections, show that the silver staining afforded by the silver diamine "fast technique" is similar to that displayed by the "multidentate ligand" plus a silver nitrate solution treatment.(ABSTRACT TRUNCATED AT 250 WORDS)
Histological Techniques↗
Identification of nucleolus organizer regions (NORs) in normal and neoplastic human cells by the silver-staining technique.
Silver nitrate has been used to demonstrate the chromosomal location of ribosomal cistrons in nine tissue-culture lines derived from human tumors of various pathological origins. Control individuals have a particular modal number (range 7--10) of D- and G-group chromosomes stained with silver. In the controls, 96.2% of the D- and G-group chromosomes that have a stalk show silver staining, while no relationship can be seen in acrocentric chromosomes without stalks. The tumor cells, whose modal chromosome numbers range from 42 to 68, possess variable numbers of acrocentrics (11--18). The number of chromosomes stained with silver, however, remained at control levels (range, 6--9). These data indicate that, in humans, silver staining may not identify all NORs that contain structural ribosomal genes.
Cell Line↗
Silver staining of proteins in polyacrylamide gels: increased sensitivity through a combined Coomassie blue-silver stain procedure.
A combined Coomassie blue-silver stain method has been developed in sodium dodecyl sulfate-polyacrylamide gels for the detection of proteins using the model compounds bovine serum albumin, lysozyme, and recombinant DNA-derived human insulin. Sensitivity was enhanced 2.2 to 8.6 times by the new method relative to that of silver staining alone. The new method may also be useful in enhancing detection sensitivities of other proteins.
Electrophoresis, Polyacrylamide Gel↗
Abundance of protein-bound sulfhydryl and disulfide groups at chromosomal nucleolus organizing regions: a cytochemical study on the selective silver staining of NORs.
Silver stainability of the chromosomal nucleolus organizing regions that contain the structural genes for ribosomal RNA can be abolished by proteolytic and oxidative treatments. Histone extraction has no effect. This indicates that reducing groups of non-histone chromosomal proteins are responsible for silver staining. Treatment with fluorescent sulfhydryl and disulfide specific reagents followed by silver staining demonstrates coincidence of silver dots and brightly fluorescent spots at the short arms of human acrocentric chromosomes where ribosomal RNA-genes are located. After treatment with cupric sulfite reagent in the presence of urea fluorescence and silver staining was no longer possible. Silver staining has been reported to be associated with ribosomal RNA-gene activity. Acrocentric chromosomes that are negative in silver staining also lack the brightly fluorescent spots. Therefore, we conclude that an abundance of protein-bound sulfhydryl and disulfide groups occur at nucleolar organizing regions with active genes. Differentially fluorescing spots could not be observed after staining with fluorescamine. So, either the sulfhydryl reagents used in this study are much more sensitive than fluorescamine to study protein distributions in cytological preparations, or our observations point to a local accumulation of some specific protein(s) rich in sulfhydryls. The presence of many sulfhydryl and disulfide groups at the nucleolus organizing regions seems suggestive of a great flexibility of protein(s) by transition of sulfhydryl groups to disulfide bridges and vice versa at these highly active regions of the genome.
Cells, Cultured↗
Effect of diethyldithiocarbamate (DEDTC) on sulphide silver stained boutons. Reversible blocking of Timm's sulphide silver stain for "heavy" metals in DEDTC treated rats (light microscopy).
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Amygdala↗