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PubMed1985

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↗

PubMed1990

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↗

PubMed1990

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↗

PubMed1988

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↗

PubMed1988

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↗

PubMed1977

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↗

PubMed1985

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↗

PubMed1979

Pattern of activity of nucleolus organizers during spermatogenesis in mammals as analyzed by silver-staining.

Silver-staining in the nuclei and chromosomes of spermatogenesis of four species of mammals (Man, Mus musculus, Rattus norvegicus, and Cavia cobaya) was investigated qualitatively and quantitatively. These species show a very similar pattern of activity of the nucleolus organizer regions (NORs) during the various stages of spermatogenesis. Silver precipitates are detectable in growing spermatogonia and up until the pachytene stage of meiotic prophase. During the meiotic metaphases I and II and during interkinesis silver-stainability disappears completely. A resumpton of silver-stainability occurs in round spermatids indicating a postmeiotic reactivation of NORs. This process does not persist beyond the early elongation phase. The quantitative determination of the silver-covered areas in relation to the total nuclear areas reveals minor differences between the species investigated with regard to the times and extents of maximum activation. The known localizations of the NORs in the karyotypes of the species investigated was confirmed using metaphase-preparations derived from somatic tissues.

Animals↗

PubMed1979

Sequential silver staining and hybridization in situ on nucleolus organizing regions in human cells.

Chromosome preparations from eight individuals were first stained with silver nitrate to reveal the nucleolus organizing regions (NORs) and then hybridized in situ with ribosomal RNA. In six individuals the size of the silver-staining regions was positively correlated with the amount of label present after hybridization in situ. Thus the variation in silver-staining intensity among chromosomes was largely explained by variation in the number of rDNA gene copies per NOR. However, in two individuals this correlation was absent, suggesting that other factors can also influence the size of the silver-staining region.

Chromosome Banding↗

PubMed1985

Opposite staining effect of two silver-staining techniques on sister chromatids.

Opposite differential staining between sister chromatids was obtained by two silver-staining techniques on chromosomes replicated twice in medium containing 5-bromodeoxyuridine (BrdU) and pretreated with Hoechst plus black light. Both silver-nitrate and silver-carbonate staining were affected by chemical extraction and enzyme digestion of chromosomal proteins. Prestaining of silver nitrate or silver carbonate also blocked the fluorescences of protein dyes. However, removal of chromosomal DNA affected the silver-carbonate but not the silver-nitrate staining; the fluorescences of DNA dyes were blocked by the prestaining of silver carbonate but not silver nitrate. Chromosomal protein labelling was released only slightly and its relative amount between BrdU bifilarly substituted and unifilarly substituted chromatids was unchanged during pretreatment of Hoechst plus black light. We speculate that chromosomal non-histones are the targets for silver-nitrate stain, and DNA-non-histone complexes for silver-carbonate stain.

Animals↗

PubMed1988

Mercury localization in mouse kidney over time: autoradiography versus silver staining.

Several methods of silver staining have been employed to localize mercury in tissue, under the assumption that the techniques represent total Hg, but recent reports have suggested that these stains are specific for a limited fraction of the Hg present in some samples. Magos et al. (1985, Arch. Toxicol. 57, 260-267) hypothesized that the stains actually vary with inorganic mercury content. The purpose of the present study was to compare localization by radiolabeling to localization by one silver stain, the photoemulsion histochemical technique, in tissues prepared to contain a range of levels of total Hg and a range of levels of inorganic Hg. Mice dosed with 8 mg Hg/kg as MeHg were killed 24 hr, 1 week, or 2 weeks after exposure, to allow a decrease in total Hg and an increase in the proportion of demethylated Hg over time. Mice dosed with 4 mg Hg/kg as HgCl2 provided samples in which all the Hg present was in the inorganic form. Atomic absorption of kidneys of mice dosed with MeHg showed that total Hg fell from 55 micrograms/g to 39 to 25 over 2 weeks, while the inorganic fraction climbed from about 2 to 27 to 35%. Grain counts from autoradiographs of 203Hg-labeled sections correlated with total Hg content at +0.88, but silver staining was correlated with inorganic Hg content, appearing only at late termination times in MeHg-exposed animals, but soon after dosing in mice exposed to inorganic Hg. The photoemulsion histochemical technique revealed a substance strictly localized in the proximal tubules, while autoradiographs and grain counts showed total Hg to be present throughout the kidney tissue. These results support the contention that silver stains are selective for inorganic Hg and suggest that the distribution of inorganic Hg, whether introduced experimentally or by gradual demethylation, is different from the distribution of MeHg. If subsequent studies support the association of silver stains with inorganic Hg, it should be possible to localize Hg in histologic sections, distinguishing between organic and inorganic forms, which differ in toxicity.

Animals↗

PubMed1990

Identification of nucleolar organizer regions in non-neoplastic and neoplastic hepatocytes by the silver-staining technique.

The silver staining technique to demonstrate nucleolar organizer region (NOR)-associated proteins (AgNORs) was applied to a variety of liver tissues, including chronic persistent hepatitis (CPH), chronic active hepatitis (CAH), liver cirrhosis (LC), liver cell dysplasia (LCD), focal nodular hyperplasia (FNH), adenomatous hyperplasia (AH) and hepatocellular carcinoma (HCC). In the present study, only discrete, easily counted black dots within nuclei and silver-stained nucleolus were counted under a magnification of x400 without oil-immersion objectives. The mean AgNOR counts of HCC and LCD were significantly higher than that of normal hepatocytes, and 77% of cases of LCD and 56% of HCC had mean AgNOR counts more than 2, whereas those in CPH, CAH, LC, FNH and AH were always less than 2 and were not different from that of normal hepatocytes. Among HCC, the mean number of AgNORs increased with the grade of the tumor. However, the AgNOR counts of grade I HCC were always less than 2 and overlapped with those of normal hepatocytes and other benign categories. All cases with mean AgNOR counts of more than 2 turned out to be HCC, except LCD which exhibited characteristic histologic appearances easily distinguished from HCC. These findings suggest that AgNORs could be quantitatively useful in evaluating the grade of HCC, even under routine microscopic examination without oil-immersion objectives, and mean AgNOR counts of more than 2 per nucleus are hallmarks of HCC.

Antigens, Nuclear↗

PubMed1985

Immuno gold staining (IGS) and immuno gold silver staining (IGSS) for the identification of the plant pathogenic bacterium Erwinia amylovora (Burrill) Winslow et al.

For the identification of the plant pathogenic bacterium Erwinia amylovora, the immuno gold staining (IGS) and immuno gold silver staining (IGSS) techniques are tested. The IGS and IGSS methods are at least as sensitive an indirect immunofluorescence and require less primary antiserum. Moreover they have the advantage that the preparations can be conserved permanently and unchanged. The preparation of the IGS can be observed with transmitted light or--with considerable better result--using epipolarization microscopy. The IGSS method deserves special attention because of its high contrast in normal brigth field microscopy with transmitted light.

Animals↗

PubMed1985

Silver staining in clinical cytogenetics.

Silver staining of human chromosomes at prometaphase or metaphase identifies variants in the stalk (nucleolar organizing) regions of acrocentric chromosomes (Nos. 13, 14, 15, 21, 22). Variants are defined by size, number, and morphology of silver staining areas. They are heritable polymorphisms and have not been associated with clinical abnormalities. However, these variants are useful in clinical cytogenetics, specifically in studies attempting to determine whether genetic material has been gained or lost in chromosomal rearrangements, the origin of chromosomal aberrations, the origin of cells in tissue culture, the chromosomal location of single genes, clonal origin of tumors, the zygosity of twins, and paternity. Some chromosomal aberrations require silver staining for their definition. Because loss of the stalk regions per se is apparently not deleterious, demonstration that chromosomal breaks occurred within this region without concomitant loss or gain of genetic material essential for normal human development provides basis for a good prognosis for the individual with the chromosomal rearrangement resulting from such breakage. The principle underlying most of the other applications is to determine whether variants being compared are identical or dissimilar, and to make inferences from these results (e.g., variants in monozygotic twins should all be identical, whereas in dizygotic twins they are as similar as in any pair of sibs). Silver staining is a valuable technique for special questions in clinical analysis.

Chromosome Aberrations↗

PubMed1988

Cytogenetic study of silver-staining NOR in 8-cell-stage mouse blastomeres fused to 1-cell-stage embryos.

Isolated blastomeres from 8- to 16-cell-stage embryos were fused by standard micromanipulatory means with either unfertilized eggs or fertilized or haploid parthenogenetically activated pronuclear-stage embryos. The hybrid eggs/embryos were incubated overnight in the presence of Colcemid until they had entered the first cleavage division. Air-dried chromosome preparations were then stained with silver nitrate in order to detect active nucleolar organizing regions (NOR). While control unfertilized eggs and 1-cell-stage fertilized and parthenogenetically activated embryos showed no evidence of silver-staining NOR-positive regions, the metaphase plates from 8- to 16-cell embryos showed characteristic NOR-positive regions, while their interphase nuclei also showed a characteristic reticular staining appearance. When hybrids between blastomere nuclei and unfertilized eggs were examined, none of the blastomere nuclei entered mitosis. However, when hybrids between blastomere nuclei and fertilized embryos were examined, in two thirds of the embryos, a single blastomere-derived diploid metaphase plate was present in association with two pronuclear-derived haploid metaphase plates. In most instances, the blastomere-derived chromosomes did not display silver-nitrate-staining NOR. Similar findings were observed when the blastomere-derived chromosomes in hybrids between blastomere nuclei and haploid parthenogenetic embryos were analysed. In the majority of cases, when blastomere nuclei remained in interphase, the characteristic silver-nitrate-staining fine reticular material either was not seen, or the nuclear contents were dispersed into clumps of chromatin-like material. Occasionally, the diploid chromosomes in the hybrids displayed morphological abnormalities. Our findings suggest that the cytoplasm of activated (but not nonactivated) 1-cell embryos is capable of influencing the nucleolar activity of the introduced 8- to 16-cell nuclei, effectively erasing from their chromosomes the memory of at least three previous rounds of rRNA synthesis.

Animals↗

PubMed1990

Application of the chromogenic reaction to conventional silver staining, the Ag-NOR staining and the silver-intensified immunogold technique.

The principle of the chromogenic reaction and the transformation of "black and white" histochemical staining results or immunohistochemical signals to coloured microscopic images is described. The chromogenic reaction was optimized and is, so far, possible with either cyan-blue or magenta-red reaction products. The application of the chromogenic reaction to conventional silver stain was optimal in the Lendrum staining resulting in red or blue stained reticulin fibres. The Ag-NOR staining of the nucleolus organizing region (NOR) could be transformed by the same reaction to coloured reaction products as well as the silver-intensified immunogold technique in immunocytochemistry.

Arthritis, Rheumatoid↗

PubMed1986

Detection of cell surface antigens in cryostat sections with immunogold-silver staining.

Immunogold-silver staining was used for the detection of lymphocyte cell surface antigens in cryostat sections of lymphoid tissues. The sections were incubated with monoclonal mouse antibodies and then with colloidal gold-labeled goat anti-mouse antibodies. They were then immersed in a physical developer, counterstained, and mounted. In light microscopy, the tissue architecture was well preserved, and a dark labeling was seen on the positive cells. Optimal labeling conditions were determined. The distribution of the lymphocyte subsets, as defined by a panel of monoclonal antibodies in tonsil and reactive lymph nodes, was similar to that found with a biotin-avidin-horseradish peroxidase method. The monoclonality of the neoplastic cells in lymph nodes of B-cell non-Hodgkin's lymphomas clearly could be demonstrated. The sensitivity of the technic was comparable with that of the biotin-avidin-horseradish peroxidase labeling method. In addition, immunogold-silver labeling was combined with acid phosphatase cytochemistry.

Antibodies, Monoclonal↗

PubMed1975

Visualization of nucleolar organizer regions im mammalian chromosomes using silver staining.

A simple ammoniacal silver staining procedure, designated Ag-AS, differentially stains the chromosomal locations of ribosomal DNA in certain mammalian species. This was critically demonstrated by Ag-AS staining of the nucleolus organizer regions in karyotypes of the same species and cell lines used for locating the ribosomal cistrons by DNA/RNA in situ hybridization. With Ag-AS, silver stained NORs (Ag-NORs) are visualized as black spherical bodies on yellow-brown chromosome arms. Ag-NORs were visualized throughout mitosis at the secondary constrictions in the rat kangaroo, Seba's fruit bat, Indian muntjac, and Rhesus monkey. The Chinese hamster and cattle have telomeric Ag-NORs, the mouse subcentromeric Ag-NORs, and the field vole Ag-NORs as minute short arms or choromosomal satellites. Ag-NORs occur at both secondary constrictions and at telomeres in the cotton rat. Variability in Ag-NOR pattern included differences in the number of Ag-NORs per cell within a cell population, size of Ag-NORs among chromosomes of a complement, and presence of Ag-NOR on particular chromosomes in two cell lines of the Chinese hamster. The available cytochemical data suggest that the Ag-AS reaction stains chromosomal proteins at the NOR rather than the rDNA itself.

Animals↗