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PubMed1993

Detection of ascorbic acid in the eye of the early chicken embryo by silver staining.

Ascorbate is found in high concentrations in the aqueous, lens and retina of a variety of animal species. Several functions have been proposed for this, including a vital role in aqueous secretion. We investigated the possible presence of ascorbate in the early embryonic chicken eye as a marker for the differentiation of the ciliary epithelium. Embryonic chicken eyes were processed for histochemical demonstration of ascorbate by silver staining. A strongly positive reaction was found in the interstitial matrix between the lens rudiment and the optic vesicle of the 2-day chicken embryo. By 3 days, with lens and optic cup separating from each other, the silver grains were concentrated in the vitreous cavity, primarily in a band-like configuration between the lens equator and the rim of the optic cup, which is the presumptive ciliary epithelium. A weaker reaction was seen in the lens vesicle and primitive neural retina. During the second and third day of incubation the optic vesicle is just beginning to expand. The appearance of ascorbic acid in the newly formed vitreous cavity at this time may indicate its importance in the biosynthesis of the matrix that is filling the expanding optic vesicle.

Animals↗
PubMed1990

A reliable method for simultaneous demonstration of two antigens using a novel combination of immunogold-silver staining and immunoenzymatic labeling.

We have developed a reliable and sensitive immunohistochemical staining technique which allows the simultaneous demonstration of two different antigens expressed in or on the same cell (referred to as mixed labeling), together with the evaluation of the general histopathological appearance of the tissue. The staining procedure combines a three-step (streptavidin-biotin) immunogold-silver staining (IGSS) with a three-step immunoenzymatic labeling. For this purpose, we investigated the compatibility of IGSS with various substrates of peroxidase or alkaline phosphatase (AP). Highly reliable and discernible mixed labeling was achieved only after initial labeling with IGSS followed by AP labeling using the substrates naphthol AS-MX phosphate/Fast Blue or naphthol AS-BI phosphate/New Fuchsin, respectively. To ensure utmost specificity, we applied FITC-conjugated mouse monoclonal antibodies and rabbit anti-FITC immunoglobulins visualized by AP-labeled immunoglobulins and the respective substrate in a final step. This novel approach provides an excellent means for demonstration of immunocompetent cells and unequivocal determination of the percentage of specific cell subsets in infiltrated tissue. The advantages of this method, as compared with double immunofluorescence or double immunoenzymatic labeling, were investigated and are discussed.

Antibodies, Anti-Idiotypic↗
PubMed1986

The analysis of lipopolysaccharide (endotoxin) in meningococcal polysaccharide vaccines by silver staining following SDS-polyacrylamide gel electrophoresis.

A simple physical-chemical method for estimating the lipopolysaccharide (LPS, i.e. endotoxin) content of meningococcal polysaccharide vaccines is described. The low molecular weight LPS was first separated from the high molecular weight polysaccharide by SDS-polyacrylamide gel electrophoresis. The LPS was then selectively visualized by a highly sensitive silver stain which can detect 5 ng of meningococcal LPS. The method can detect concentrations of LPS in meningococcal polysaccharides as low as 0.01%, and was used to measure semiquantitatively the LPS in bulk powders of meningococcal polysaccharides and in vaccines in final containers. The LPS in the bulk powders and in the vaccines was also assayed by the gelation of Limulus polyphemus ameobocyte lysate and the results of the two methods were in agreement.

Animals↗
PubMed1996

Multiplex sets for the amplification of polymorphic short tandem repeat loci--silver stain and fluorescence detection.

Multiplex PCR amplification systems were developed using well-characterized, polymorphic short tandem repeat (STR) loci. Eight loci utilized in the multiplex amplifications included HUMCSF1PO, HUMTPOX, HUMTH01, HUMVWFA31, HUMF13A01, HUMFESFPS, HUMBFXIII and HUMLIPOL. From this list, three or four non-overlapping loci were simultaneously amplified, separated by denaturing polyacrylamide gel electrophoresis and visualized using silver stain or fluorescence detection. The multiplex PCR amplification systems offer a non-isotopic method for rapid, simple and accurate analysis of STR loci. This high-throughput method for DNA identification has immediate and valuable application in forensic analysis, paternity determination, tissue culture strain identification and bone marrow transplantation studies.

DNA↗
PubMed1998

The use of epithelial membrane antigen and silver-stained nucleolar organizer regions testing in the differential diagnosis of mesothelioma from benign reactive mesothelioses.

BACKGROUND: The accurate diagnosis of pleural lesions obtained from small closed biopsy is difficult. As yet there is no single reliable test to distinguish between malignant and benign mesothelial tissue. METHODS: Immunostaining of epithelial membrane antigen (EMA) and the quantitation of silver stained nucleolar organizer regions (AgNORs) each were applied to benign and malignant histologic sections of pleural and peritoneal biopsies. The usefulness of these stains was tested both individually and in combination in the diagnosis of epithelial malignant mesothelioma. RESULTS: One hundred and three of the 141 malignant lesions (73%) were immunoreactive for EMA but only 3 of the 73 benign lesions (4%) reacted equivocally, and none positively. The average count of AgNORs/cell in malignant lesions (n = 80) was elevated compared with benign cases (n = 26), but a significant overlap was exhibited in the AgNOR count and this form of analysis was considered to be of little value in distinguishing benign from malignant mesothelial processes. Much less overlap was observed when the average AgNOR area was measured. By using the maximum benign AgNOR area of 0.6677 microm2 as the upper threshold, 51 cases (63.8%) were identified as malignant; the test demonstrated 100% specificity and 63.8% sensitivity. By combining the EMA and AgNOR results, 76 of 80 of the malignant mesothelioma cases (95%) tested positive for at least 1 of the tests with no false-positive results identified. CONCLUSIONS: This study confirms the usefulness of EMA in diagnosing malignant and benign mesothelial lesions, and demonstrates the enhanced diagnostic value of combining EMA immunoreaction with the average area of AgNOR per cell, thereby increasing sensitivity in the diagnosis of epithelial malignant mesothelioma.

Adolescent↗
PubMed1992

Changes in nucleolar transcriptional activity in hepatitis B virus-associated chronic liver diseases. Preliminary results from a quantitative study of silver-stained nucleoli.

Modifications of gene expression may occur in hepatitis B virus (HBV)-related chronic liver diseases, possibly also involving ribosomal RNA (rRNA) genes contained in the nucleolus. Changes in the level of transcriptional activity of rRNA genes are reflected by variations in the number and/or size of the nucleoli. Therefore a quantitative analysis of the silver-stained nucleoli (AgNus) was performed in a small series of liver needle biopsies from patients with HBV+ chronic persistent hepatitis (CPH) (n = 3), HBV+ chronic active hepatitis (CAH) (n = 3) and HBV+ cirrhosis (CIR) (n = 3). In each case, 100 hepatocytes were selected. The number of the nucleoli (AgNuN), their total area (tAgNuA), the average area of each nucleolus (xAgNuA), the nuclear area (NA) and the percentage ratio of tAgNuA related to NA (rAgNuA) were determined for each hepatocyte nucleus. The pooled mean values of all the features were significantly different (p less than 0.001) among the case groups. The results point towards a remarkable increase of nucleolar activity in CAH in comparison with CPH, whereas an additional increment of this activity is associated with the progress from CAH to CIR.

Biopsy, Needle↗
PubMed1984

Purification of retinal S-antigen to homogeneity by the criterion of gel electrophoresis silver staining.

This report describes a procedure by which high performance liquid chromatographic (HPLC) techniques are used to obtain homogeneous S-antigen. Conventionally prepared S-antigen was purified further by fractionation on the anion exchange Mono-Q column followed by gel filtration on a TSK-3000 column. This procedure produced an S-antigen preparation, which appeared homogeneous by gel electrophoresis using the very sensitive silver staining method. The purified material retained its immunochemical and uveitogenic activity. The availability of homogeneous S-antigen will facilitate studies aimed at elucidating, at the molecular level, the mechanism by which S-antigen induces uveitis.

Animals↗
PubMed1986

Combined alcian blue and silver staining of glycosaminoglycans in polyacrylamide gels: application to electrophoretic analysis of molecular weight distribution.

Oligomeric and polymeric fragments of glycosaminoglycans may be separated for rapid analysis by electrophoresis through a 10% polyacrylamide matrix. A ladder-like series of bands is observed, in which adjacent major bands correspond to species differing in chain length by one disaccharide unit. The component species are detected by a combined alcian blue and silver staining protocol. Detection limits are less than 50 ng per band, or approximately 2-5 micrograms total load for polydisperse samples. Densitometry of the stained gel may be used to determine molecular weight averages and distribution. The applicable molecular weight ranges are approximately 4000 to 100,000 for hyaluronate, or 1500 to 40,000 for chondroitin and dermatan sulfate samples of moderate charge density heterogeneity.

Alcian Blue↗
PubMed1988

Development of polyacrylamide gels that improve the separation of proteins and their detection by silver staining.

Background staining that is associated with silver detection of proteins and nucleic acids in polyacrylamide gels has been shown to be due mostly to the amide groups in methylenebisacrylamide, a commonly used gel crosslinker. In attempts to reduce this background staining, eight existing crosslinking agents were tested. All of these proved to be unsuitable. Six new crosslinking agents were synthesized and tested. Of these, diacrylylpiperazine provided increased physical strength, improved electrophoretic separation of proteins, and silver staining detection of proteins with reduced background stain.

Acrylamides↗
PubMed1990

Distinctive, rapid, and easy labeling of diffuse plaques in the Alzheimer brains by a new methenamine silver stain.

We have developed a new methenamine silver (MS) stain for detecting diffuse plaques distinctively on paraffin-embedded tissue sections of Alzheimer-type dementia, Down's syndrome, and mentally normal aged brains. This rapid and easy method selectively labels amyloid-related component of senile plaques, but not of kuru plaques found in Gerstmann-Sträussler syndrome. Our MS stain shows almost the same staining pattern as that of the beta protein immunostaining with formic acid pretreatment. Therefore, new MS stain is appropriate to routine or screening studies for senile plaques including diffuse plaques.

Alzheimer Disease↗
PubMed2003

Visual gene diagnosis of HBV and HCV based on nanoparticle probe amplification and silver staining enhancement.

A visual gene-detecting technique using nanoparticle-supported gene probes is described. With the aid of gold nanoparticle-supported 3'-end-mercapto-derivatized oligonucleotide serving as detection probe, and 5'-end -amino-derivatized oligonucleotide immobilized on glass surface acting as capturing probe, target DNA was detected visually by sandwich hybridization based on highly sensitive "nano-amplification" and silver staining. Different genotypes of Hepatitis B and C viruses in the serum samples from infected patients were detected using home-made HBV, HCV, and HBV/HCV gene chips by the gold/silver nanoparticle staining amplification method. The present visual gene-detecting technique may avoid limitations with the reported methods, for its high sensitivity, good specificity, simplicity, speed, and cheapness. This technique has potential applications in many fields, especially in multi-gene detection gene chips coupled with the detection will find applications in clinic. Additionally, resonance Rayleigh light scattering (RLS) spectroscopy is used, for the first time, to judge and monitor the immobilization of gene probes on gold nanoparticle surfaces.

DNA Probes↗
PubMed1999

Potentially malignant and malignant lesions of the lip. Role of silver staining nucleolar organizer regions, proliferating cell nuclear antigen, p53, and c-myc in differentiation and prognosis.

The cellular changes leading to carcinoma of the lip are still not completely understood. This study was carried out on 44 malignant and potentially malignant lesions of the lower lip [30 squamous cell carcinomas (SCC), 7 actinic cheilitis, 3 leukoplakias, and 4 nodal metastases from lower lip SCC]. Silver-stained nucleolar organizer regions (AgNORs) and the immunohistochemical expression of proliferating cell nuclear antigen (PCNA), p53, and c-myc were evaluated on formalin-fixed, paraffin-embedded sections. The results indicate that the size and numbers of AgNORs and the percentage of PCNA-positive cells are sensitive parameters for discriminating between potentially malignant lesions and SCC, and for the prognostic sub-typing of lower lip SCC. Furthermore, while p53 positivity was found more frequently in high-grade carcinomas, p53-positive cellular clones were also found in some potentially malignant lesions, a finding probably related to ultraviolet-related cellular damage. These p53-positive lesions could be considered at higher risk of progression to malignancy than the p53-negative ones, although there is no evidence for this as yet. c-myc positivity was found only in some high-grade carcinomas and metastases, and appeared correlated with the later phases of lip carcinogenesis. The combined evaluation of the proliferation status, together with the changes in p53 and c-myc oncoproteins, might constitute useful markers for the prognostic evaluation of potentially malignant, as well as malignant, lesions of the lip.

Adolescent↗
PubMed1986

[Advantages and disadvantages of silver staining of proteins in polyacrylamide gel].

Sensitivity of protein staining with Serva blue G-250 (Coomassie brilliant blue G-250 analogue) in polyacrylamide gel was determined. It has been shown that protein staining with 0.1% Serva blue G-250 results in the recovery of 80 to 35 ng of single protein, that is almost 10 times higher than reported previously for Coomassie brill. blue G-250 (or R-250) staining. The comparison of the sensitivity of Serva blue G-250 protein staining in PAAG and AgNO3 has shown that AgNO3 staining was approximately 18-30 (but not 100 times, as it had been thought before) times more effective for the majority of proteins under study. Silver staining of some proteins, for instance ribonuclease and a number of retrovirus-specific structural proteins, was of lower efficacy. Thus, to obtain reliable results protein electrophoresis in PAAG should be followed by both staining procedures.

Electrophoresis, Polyacrylamide Gel↗
PubMed1985

Sensitive colloidal metal (gold or silver) staining of protein blots on nitrocellulose membranes.

A sensitive staining method for protein blots on nitrocellulose membranes is described and compared with commonly used dye staining methods. It uses colloidal metal sols (gold or silver) stabilized with Tween 20 and adjusted to pH 3. It is based on the selective high-affinity binding of colloidal metal particles to the proteins and produces a red-purplish color (gold) or dark grey (silver). The sensitivity of this new staining method is in the same range as silver staining of polyacrylamide gels and matches the sensitivity of overlay assays. It will therefore be a useful tool for correlating the position of bands or spots of proteins detected with overlay assays with the complete electropherogram in a duplicate protein blot.

Animals↗
PubMed1990

Immunogold-silver staining method for light and electron microscopic detection of lymphocyte cell surface antigens with monoclonal antibodies.

We used the immunogold-silver staining method (IGSS) for detection of lymphocyte cell surface antigens with monoclonal antibodies in light and electron microscopy and compared this procedure with the immunogold staining method. Two different sizes of colloidal gold particles (5 nm and 15 nm) were used in this study. Immunolabeling on cell surfaces was visualized as fine granules only by IGSS in light microscopy. The labeling density (silver-gold complexes/cell) and diameters of silver-enhanced gold particles on cell surfaces were examined by electron microscopy. Labeling density was influenced not by the enhancement time of the physical developer but by the size of the gold particles. However, the development of shells of silver-enhanced gold particles correlated with the enhancement time of the physical developer rather than the size of the colloidal gold particles. Five-nm gold particles enhanced with the physical developer for 3 min were considered optimal for this IGSS method because of reduced background staining and high specific staining in the cell suspensions in sheep lymph. Moreover, this method may make it possible to show the ultrastructure of identical positive cells detected in 1-micron sections counterstained with toluidine blue by electron microscopy, in addition to the percentage of positive cells by light microscopy.

Animals↗
PubMed1991

Re-evaluating silver-stained nucleolar organizer regions (AgNORs) in problematic cutaneous melanocytic lesions: a study with quantitation and pattern analysis.

Previous studies enumerating AgNORs in cutaneous melanocytic lesions have produced inconsistent results. It is probable that such inconsistencies arise from differences in fixation, staining technique, and counting strategies. Our group, having demonstrated an improved method of silver staining and having optimized counting, is now able to reconsider the role of AgNORs in evaluating borderline melanocytic lesions. Diagnostic problems similar to those encountered in routine practice have been examined. It is shown that only by counting intra- and extranucleolar AgNORs in combination with assessing the pattern of AgNOR dispersal is it possible to (1) discriminate dysplastic naevi from melanoma and (2) distinguish Spitz naevi and pigmented spindle cell naevi from melanoma. An analysis of AgNOR numbers alone results in considerable overlap between the groups studied. The value of assessing patterns of AgNOR dispersal within and outside clustered nucleolar structures is emphasized. Lesions labelled as minimal deviation melanoma (by experts in dermatopathology) were also investigated. The majority of these specimens form a distinct group lying apart from control naevi and melanomas. This finding, whilst of interest, is difficult to evaluate because of poorly defined diagnostic criteria.

Cytological Techniques↗
PubMed1980

Comparison of N banding and silver staining of human NORs.

A comparison of the sensitivity of silver and Giemsa N band staining for detection of the human nucleolar organizer regions (NORs) was carried out in cultured lymphocytes from three unrelated individuals. In sequentially stained cells examined from each subject, the mean number of Ag-NORs was greater than the mean number of N bands. The difference in stainability between the two techniques was shown to be highly significant (chi 2 = 430.2; P less than 0.01) when 100 cells pooled from all individuals were analyzed. These results suggest a greatly reduced sensitivity of the NOR for Giemsa, as opposed to silver. This conclusion is also supported by the finding that slides aged for over 1 week did not N band, while silver staining was accomplished on slides of any age. Several possible factors that might be responsible for the difference in staining response are discussed.

Chromosome Banding↗