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PubMed1982

Variations in the activity of nucleolar organizers in different tissues, demonstrated by silver staining of human normal and leukemic cells.

A simple silver-staining technique that demonstrates those nucleolar organizing regions of metaphase chromosomes which are transcriptionally active during the preceding interphase (AgNORs) has been applied to cells obtained from the bone marrow and mitogen-stimulated peripheral blood lymphocyte cultures of hematologically normal individuals and patients with various forms of leukemia. In the majority of bone marrow cells from the normal controls and many of the patients, the number of cells with detectable AgNORs, and the staining intensities in those cells which were Ag+, were markedly reduced compared with the levels found in blood lymphocytes. The numbers of cells having satellite associations and the numbers of chromosomes participating in these associations also generally reflected the proportions of AgNORs present. When patterns of bone marrow silver staining were compared between patients with leukemia, distinct differences were found which could be correlated with cytology. It is suggested that different cell types have characteristic AgNOR staining profiles, reflecting specific regulation of ribosomal RNA synthesis in particular cell lineages. AgNOR staining may indicate, therefore, the predominant cell types that divide in the bone marrows of patients with different forms of leukemia.

Bone Marrow Cells↗
PubMed2001

Uveal melanoma: mean of the longest nucleoli measured on silver-stained sections.

PURPOSE: To summarize and compare the various histologic methodologies for using nucleoli to assess the malignant potential of uveal melanoma. METHODS: This was an observational series of 100 samples of uveal melanoma in which histologic sections were studied. The cases were selected so that approximately half (n = 49) of the tumors were from patients who had died of metastatic malignant melanoma. The 51 remaining tumors were from patients who had survived at least 9 years without development of metastasis. Central sections from the uveal melanomas were stained using the colloidal silver nitrate stain for nucleolar organizing regions (AgNOR). These were compared with an adjacent hematoxylin and eosin (H&E)-stained section. A light microscope with a micrometer inset into the eyepiece (x10) was used at a final magnification of x1000 under oil immersion to measure the length of the nucleolus along the longest axis and the width perpendicular to that axis. From at least twenty cells selected from random fields throughout the tumor, the mean of the 10 longest and widest nucleoli (MLN) was calculated. Seven samples had to be discarded because the nucleoli were unmeasurable. RESULTS: T-tests and Cox proportional hazard regression analysis indicated that the MLN of nucleolar length as measured on AgNOR-stained slides was as significant as cell type but was more significant than other histopathologic prognosticating variables measured and evaluated in this study. These prognosticators included tumor size, calculated as the largest tumor dimension; MLN width; and MLN length, as measured on H&E-stained sections. CONCLUSIONS: It has previously been demonstrated that AgNOR-stained nucleoli, unlike H&E-stained nucleoli, can be captured and measured by an automated image analyzer with prognostically significant results. This new method of simple oil-immersion measurements of the longest AgNOR-stained nucleoli length in microscopic sections of uveal melanoma provides an inexpensive and highly significant method for predicting outcome in patients with uveal melanoma. Because of the high contrast with the background, the silver-stained nucleoli clearly define the nucleolar boundaries, rendering them readily discernible and allowing greater ease and speed of measurement when compared with H&E-stained nucleoli. The method of random sampling that was used was comparable with linear sampling in predicting outcome. Highly necrotic tumors, however, had to be excluded from the study because of loss of nucleolar morphology.

Cell Nucleolus↗
PubMed1991

Demonstration of the nucleolar organizer region by silver staining (AgNOR method) in research and in histopathological practice.

The silver-staining method for the detection of nucleolar organizer regions (AgNOR) was tested on a large material and the AgNORs evaluated in normal and pathological tissues, especially in various benign and malignant neoplasms. Pathological lesions were represented by dysplasias and in situ carcinoma of the uterine cervix, dysplasias and various histological forms of breast carcinoma, carcinomas of the lung, of urinary bladder, of prostate, various neoplasms of the gastrointestinal tract, trophoblastic disease, naevocellular naevi and melanomas of the skin, myeloproliferative diseases and haematological malignancies, and also by several cases of xenotransplanted human neoplasms in athymic nude mice. The AgNORs were evaluated using several procedures. Marked AgNOR heterogeneity in individual malignant tumours was found to be a common feature suggesting the presence of cellular subpopulations or clones with different proliferative activity and metabolic properties. Close correlation between the proliferative behaviour and the quantitative and qualitative change of AgNORs was found in most obviously malignant neoplasms. The method, however, could not recognize neoplastic cells as such and is therefore not a specific oncological marker. This limits the diagnostic contribution of the silver staining method in diagnostic pathology, where it does not exceed the value of routinely stained slides of good quality. In spite of these limitations, the AgNOR staining method seems to represent a valuable contribution in the study of some aspects of the proliferative lesions, such as their proliferative and metabolic activity, hormonal dependence etc.

Animals↗
PubMed1995

Spectrophotometric quantitation of silver in X-ray film images and silver-stained electrophoresis gel bands.

This paper describes a novel spectrophotometric method for quantitating the silver in silver-stained electrophoresis gel bands and X-ray film images. Silver in excised areas is quantitated by (i) oxidation/solubilization of Ag0 to Ag+ with nitric acid, (ii) neutralization followed by extraction into an organic solution of dithizone (diphenylthiocarbazone), and (iii) visible absorbance measurement at 600 nm. Spectrophotometric quantitation is possible because dithizone undergoes a dramatic color change from blue-green to yellow upon binding Ag+ (delta epsilon 600 = 3.28 x 10(4) M-1 cm-1). Experiments with silver-stained gels and with X-ray film images obtained by chemiluminescence and autoradiography demonstrated that the color change relates to the amount of silver present and hence to the amount of material represented by the band or image. This method is a convenient, inexpensive alternative to the use of a densitometer to quantitate silver-containing images.

Electrophoresis, Polyacrylamide Gel↗
PubMed1994

Number and size of silver-stained nucleoli (Ag-NOR clusters) in canine seminomas: correlation with histological features and tumour behaviour.

Twenty spontaneous canine seminomas were examined. A statistically significant difference (P < 0.01) was detected in both the mean silver-stained nucleolar area/cell and the mean silver-stained nucleolar number/cell between (1) two intraductal seminomas with no signs of invasion, and (2) three intraductal ones with signs of invasion and 15 diffuse-type seminomas (two of which metastasized 3 and 12 months after surgical treatment). Furthermore, the metastasizing seminomas showed a mean silver-stained nucleolar area/cell which was significantly greater than that observed in all the other seminomas examined (P < 0.01). It is concluded that quantitative studies of nucleolar changes are useful in evaluating the biological behaviour of spontaneous canine seminomas.

Animals↗
PubMed1984

The potential of the immunogold-silver staining method for paraffin sections.

The immunogold-silver staining technique is shown to be of great value in the detection of regulatory peptide-containing nerves and endocrine cells in routinely fixed, paraffin-wax-embedded tissues. The method appears to be better for this system than peroxidase anti-peroxidase (PAP) which can yield poor or variable results. Antibodies to regulatory peptides, including calcitonin gene-related peptide (CGRP), substance P, neuropeptide tyrosine (NPY), glucagon, pancratic polypeptide, and somatostatin 14 and 28, as well as to neurofilaments, neuron-specific enolase (NSE) and S-100, were used on sections of a variety of tissues from rat and pig including respiratory tract, skin, gut, pancreas, vagina, uterus, fallopian tube and kidney. In all cases, stronger immunostaining of nerves was obtained with the immunogold-silver technique than with PAP. The inherent density of the staining was also found to improve the visibility of endocrine cells in the section, and to permit the use of routine histological stains for counterstaining. As immunogold-silver staining is sensitive, rapid, cheap and avoids hazardous reagents, we feel it has great potential for the immunostaining of nerves and endocrine cells that contain regulatory peptides in routinely fixed and embedded tissues and may prove useful in pathology.

Animals↗
PubMed1988

Improved silver staining procedure for fast staining in PhastSystem Development Unit. I. Staining of sodium dodecyl sulfate gels.

A new modification of silver staining of proteins in sodium dodecyl sulfate polyacrylamide gels is adapted to automated staining in PhastSystem Development Unit. The use of a reduction step, after fixation, with thiosulfate in alcoholic sodium acetate buffer results in a considerable increase in sensitivity without the need for a recycling step. The detection limit is tenfold lower than in the silver staining procedure recommended so far for PhastSystem and corresponds to 0.05-0.1 ng protein per band. Total staining time with the new procedure is 75 min.

Alkylation↗
PubMed1992

Electron microscopic study of paired helical filaments and cerebral amyloid using a novel en bloc silver staining method.

A one step en bloc silver staining method which was originally established to study nucleolar organizer regions has been applied for the demonstration of both paired helical filaments (PHF) and extracellular cerebral amyloids in semi-thin sections and at the electron microscopic level. The three forms of PHF can be visualized: (1) neurofibrillary tangles are shown in all stages from first appearance in form of intracellular patches of PHF to severely degenerated shadow-like "ghost" tangles; (2) neuropil threads are distinctly stained in great numbers; and (3) PHF are easily detected as neuritic components in amyloid plaques. All forms of fibrillar extracellular amyloid structures, i.e. "diffuse", "classical" and "burnt out" plaques, are well demonstrated; congophilic angiopathy reveals amyloid preferentially in arteries and arterioles of the leptomeninges and cortex ranging from small circumscribed patches to large circumferential amounts with occasional plaque-like condensations or broad loose accumulations of amyloid; perivascular cuffs and laminar subpial deposits of amyloid are stained as well. At the electron microscopic level all lesions are clearly visible in non uranyl/lead-stained specimens, characterized by varying numbers of silver grains on a pale background. The detailed demonstration of structures in archival material, which had been stored in paraffin and re-embedded for electron microscopy, is due to the demonstration of argyrophilic structures by the protective colloidal developer of gelatin and formic acid and to the proteolytic resistance of insoluble PHF and extracellular amyloids in plaques and congophilic angiopathy.

Alzheimer Disease↗
PubMed1977

Silver staining the caveolae intracellularis of smooth muscle.

Bodian silver staining of guinea-pig Taenia coli produces a regular distribution of silver at the periphery of the smooth muscle cells. Electron microscopic analysis of this precipitate revealed that the membrane of the caveolae intracellularis was the only structure at the periphery of the cells that consistently exhibited a high affinity for silver. Thus, this staining method demonstrates that caveolae are distributed over most of the cell surface in an orderly pattern.

Animals↗
PubMedDate not supplied

Simultaneous visualization of rDNA clusters and the nucleolus by combining fluorescence in situ hybridization and silver staining.

We designed two procedures to visualize simultaneously clusters of ribosomal RNA genes (rDNA) and the nucleolus in plant cells. The procedures combine fluorescence in situ hybridization (FISH) to visualize the rDNA clusters and silver staining to observe the nucleolus. When FISH is followed by silver staining, many minute FISH signals are localized in the nucleolus, and several large FISH signals are seen on the nucleolar periphery. When FISH was applied to the specimens with silver nitrate staining, large FISH signals were visualized in the nucleoplasm associated with the nucleolar periphery, but no signals were seen in the nucleoli. Thus, the two combinations of FISH and silver staining provided different details regarding the arrangement of rDNA clusters in the nucleolus of plant cells.

Allium↗
PubMed1988

Comparison of diazo-coupling, formazan, and silver staining techniques for visualizing alkaline phosphatase isoenzymes after electrophoresis in homogeneous-pore and gradient-pore polyacrylamide gels.

Three techniques for visualization of alkaline phosphatase after polyacrylamide-gel electrophoresis are compared. These are diazo-dye simultaneous coupling with the substrate sodium naphthyl phosphate and 5-chloro-2-toluene diazonium chloride; formazan precipitation with the substrate 5-bromo-4-chloro-3-indolyl phosphate and 3-[4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide; and silver staining with the substrate sodium glycerophosphate. Each staining technique was tested with gradient-pore and homogeneous-pore acrylamide-gel electrophoresis. The main factors assessed are sensitivity; separation of the human serum alkaline phosphatase isoenzymes of the liver, bone, and intestinal types; and differences in substrate affinity, as well as the complexity of each technique. Using the three techniques only minor differences in substrate affinity are evident. There is some nonspecific staining with the diazo-coupling technique but not with the formazan and silver staining techniques. The differences, in the mobility of the liver, bone, and intestinal isoenzymes achieved by homogeneous-pore gel electrophoresis are sufficient to allow them to be clearly distinguished. However, only very small differences in mobility are found with gradient-pore gel electrophoresis, but the sharper bands in this medium allow much smaller amounts of activity to be detected. As little as 160 microU of enzyme can be visualized by the diazo technique. Silver staining gives an approximately fourfold increase in sensitivity over the formazan technique, which in turn gives a fourfold increase over the diazo technique. An important aspect of the silver staining technique is the potential of increasing sensitivity much further by improvements in the photographic physical development stage.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed1997

Standardized demonstration of silver-stained nucleolar organizer regions-associated proteins in archival oral squamous cell carcinomas and adjacent non-neoplastic mucosa.

Standardized silver staining and quantitation of nucleolar organizer regions-associated proteins (AgNORs) on routinely formalin-fixed and paraffin-embedded tissues has been recently established. The present study was designed to assess its applicability and reproducibility on archival oral squamous cell carcinoma specimens, with special emphasis on the invasive tumor front. Standardized image cytometric AgNOR analysis was performed at the invasive zone and central parts of the tumors as well as in adjacent dysplastic and normal oral mucosa by applying a modified silver staining on routinely processed archival tissues after wet autoclave pretreatment for protein retrieval. A statistically highly significant and reproducible difference of all of the four standardized AgNOR parameters evaluated (mean AgNOR number, mean AgNOR area, coefficient of variation (CV) of AgNOR number, and CV of AgNOR area per nucleus) was observed between the different areas assessed; there was a statistically significant gradual decrease in the CV of the AgNOR number from the invasive tumor front to central parts of the carcinomas, from dysplastic mucosa to normal epithelium. The observed increase of AgNORs at the invasive front of oral squamous cell carcinomas seems to indicate a subgroup of tumor cells with increased biosynthetic activity and malignant potential. Wet autoclave pretreatment before silver staining of AgNORs is necessary for a reliable visualization of single AgNOR dots. Second-order statistics of AgNOR features, e.g., CV values, offer a potent and highly reproducible parameter for AgNOR quantitation.

Biomarkers, Tumor↗
PubMed1997

Dual silver staining to characterise Helicobacter spp. outer membrane components.

Helicobacter pylori is a bacterial pathogen, estimated to infect half the world's population. The bacterium is the aetiological cause of gastritis, the common precursor for peptic ulcer disease and gastric cancer. Immunisation of at-risk individuals is the most cost-effective means of dealing with such a widespread pathogen. Potential vaccine candidates need to be identified and characterised. Conventional silver staining is commonly used for the sensitive detection of bacterial protein components separated by SDS-PAGE. Modified silver stains employing periodate oxidation have also been developed for the analysis of purified bacterial lipopolysaccharide. By using these methods in parallel, as a dual silver stain, bacterial fractions can be characterised in terms of protein and LPS content. Strain differences can also be readily identified by comparing protein and LPS profiles. When combined with differential immunoblotting, the dual silver stain is a useful analytical tool for characterising potential vaccine candidate antigens.

Animals↗
PubMed1989

Differential staining of tick chromosomes: techniques for C-banding and silver-staining and karyology of Rhipicephalus sanguineus (Latreille).

A new karyotypic procedure is described for tick chromosomes along with modifications of accepted techniques for C-banding and silver-staining. The staining procedures produce patterns with staining limited to the centromeric regions and the nucleolar organizer regions (NORs). The possible reasons for the concordant patterns are discussed based on the staining characteristics of the 2 techniques. The karyotype of Rhipicephalus sanguineus possesses 20 acrocentric autosomes with a single pair of NORs located proximal to the centromere on the largest autosomal pair. The sex-determining system is XX:XO with the X chromosome being the largest chromosome in the complement and subtelocentric.

Animals↗
PubMed1990

Silver-stained nucleolar organizer regions in the differentiation of prostatic hyperplasia, intraepithelial neoplasia, and adenocarcinoma.

Nucleolar organizer regions (NORs) are loops of chromosomal DNA that ultimately direct the development of the nucleolus. These NORs are associated with argyrophilic acidic nonhistone proteins which have allowed the demonstration of NORs by a simple silver stain. This staining technique has been used to examine the number of silver-stained NORs (AgNORs) in malignant and benign conditions, and, in general, AgNOR numbers are increased in malignant tissues when compared with normal or benign conditions. More recently, this technique has been applied to premalignant lesions with varying results. We applied this silver stain to prostate lesions where nucleolar features are of diagnostic importance. The stain was applied to ten cases of prostatic hyperplasia, ten cases each of prostatic intraepithelial neoplasia (PIN) grades 1 through 3, and ten cases each of low grade, intermediate grade, and high grade adenocarcinoma. The counting was performed by image analysis. A significant mean difference did exist between low grade and the two higher grades of adenocarcinoma. There was no significant difference in AgNOR counts per nucleus in hyperplasia, PIN, or adenocarcinoma or between the three grades of PIN. The AgNOR silver stain is not useful in the differentiation of hyperplasia, the three grades of PIN, and adenocarcinoma. Also because of overlap among the three grades of adenocarcinoma, it is not useful in the differentiation of these lesions.

Adenocarcinoma↗
PubMed1990

Silver stain for ringed sideroblasts. A sensitive method that differs from Perls' reaction in mechanism and clinical application.

A modified silver stain is described for the demonstration of ringed sideroblasts in bone marrow. It is more sensitive than Perls' reaction for that purpose, especially when iron stores of marrow are low or absent. Ringed sideroblasts may still be demonstrable by silver stain in cases of sideroblastic anemia without ringed sideroblasts, for which severe iron deficiency prevents detection of the abnormal sideroblasts by Perls' reaction. As iron has been reported to be present in mitochondria of ringed sideroblasts in the form of ferric phosphate, it is possible that the silver stain demonstrates the phosphate moiety and not the iron, thus explaining its greater sensitivity in iron deficiency as compared to Perls' reaction. Further study is necessary to confirm the staining mechanism, to elucidate the composition of iron deposits, and to explain the pathophysiology of sideroblastic anemia.

Anemia, Sideroblastic↗
PubMed1984

The application of the nucleolar organizer region silver staining (AgNOR) to backscattered electron imaging (BEI).

Until recently scanning electron microscopes were mainly used to observe surfaces. However, it has been proved that a backscattered electron detector can give an image (BEI) of the specimen's internal structure after heavy metal staining. In this paper, we report how we have applied the silver staining for NOR-associated proteins to scanning electron microscopy, studying C3H10T1/2 cells in culture. This technique allows to localize, inside the nucleus, the nucleolar arrangement of AgNOR-associated proteins. In BEI imaging, the silver staining shows several intranucleolar silver spot-like deposits sometimes associated in "doublets" as on metaphasic chromosomes. These silver grains probably represent the fibrillar centre location, thought to be the interphasic counterpart of the NORs. However, these silver spot granules are more numerous during interphase.

Animals↗
PubMed1992

Identification of the plant nucleolonema using silver staining of serial ultrathin sections.

A simple silver staining technique was applied to serial ultrathin sections of Vicia faba root tip meristems to investigate the structural organization of the nucleoli. The procedure consists of three steps: hydrophilic Lowicryl resin embedding, preparation of ribbons of serial ultrathin sections, and silver impregnation. This technique provided good contrast for a filamentous structure, previously called the "nucleolonema", in nucleoli at the light microscope level. Silver appears to react with some acidic proteins involved with ribosomal RNA transcription, since the major constituents of the nucleolonema are believed to be active rRNA transcriptional units. Tracing the nucleolonema through serial ultrathin sections strongly suggests that the nucleolonema is meandering and partly coiling within the nucleolus. The present technique permits identification of the nucleolonema in other plant species and investigation of its three-dimensional structural organization.

Cell Nucleolus↗