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PubMedDate not supplied

[Comparison of the methenamine silver staining, direct fluorescent antibody and nested-polymerase chain reaction methods in the diagnosis of Pneumocystis carinii pneumonia].

Pneumocystis carinii is one of the most common causative agents of pneumonia in immunocompromised patients, but the problems in the laboratory diagnosis of the disease frequently leads to diagnosis according to the response to medical treatment. In this study, the presence of P. carinii was investigated in immunocompromised patients who were presenting with the clinical symptoms of atypical pneumonia, by Gomori methenamine silver staining (GMS), direct fluorescent antibody (DFA) test and nested-polymerase chain reaction (nPCR) methods. Fifty-three samples of 49 patients were included in the study. Twelve of the samples (22.6%) were found to be positive by nPCR, 6 of them (11.3%) were found to be positive by DFA, while only one of them (1.8%) was positive by GMS staining method. As a result, for the appropriate treatment and prophylaxis of P. carinii infections, PCR which is a rapid and reliable diagnostic test should be used for diagnosis.

DNA, Fungal↗
PubMed2006

Dysbacteriosis in silver-stained cervical smears of Dutch-Moroccan immigrants: HPV infection and preneoplasia.

The vaginal/cervical smears of a group of Moroccan immigrants were used to compare vaginal dysbacteriosis (i.e., a bacterial population change with a decrease in lactobacilli and an increase of coccoid bacteria in vaginal/cervical smears) with Dutch women. From our archives, 779 smears from Moroccan immigrants were compared with 1,060 smears of age-matched Dutch women. For bacterial flora, Jones-Marres silver stains were used to define four groups. Koilocytosis and cervical intraepithelial neoplasia (CIN) were also recorded. The bacterial vaginal flora of Moroccan immigrants and Dutch women was different. The Moroccan women had a lower dysbacteriosis than Dutch women (3% vs. 24%). Koilocytosis and CIN were less frequent in the immigrant population. The possible synergy of a disturbed vaginal flora with human papillomavirus (HPV), HIV, or cervical preneoplasia indicates that vaginal hygiene and a normal flora may have positive effects on the uterine cervix.

Case-Control Studies↗
PubMed1993

Neural cell adhesion molecule expression and clinical features in small cell lung cancer: a semi-quantitative immunohistochemical approach using an immunogold-silver staining method.

The neural cell adhesion molecule (NCAM) is a family of cell surface sialoglycoproteins mediating homotypic and heterotypic cell-cell adhesion. In tumors, NCAM is supposed to be involved with the malignant features characterized by invasive growth and metastasis. In the present study, we evaluated the correlation between NCAM expression of tumors obtained from small cell lung cancer (SCLC) patients and the clinical outcome. NCAM expression was determined semi-quantitatively by an immunogold-silver staining method using the SCLC cluster 1 monoclonal antibody NCC-LU-243. Of 20 SCLC patients studied, six patients with tumors with high NCAM expression had a poor response to chemotherapy, and a short disease-free (p = 0.011) and overall (p = 0.003) survival as compared with 14 patients having tumors with low NCAM expression. These findings indicate that the therapeutic outcome of SCLC may be partly predicted by determining the NCAM expression of the tumor.

Adult↗
PubMed1995

Application of dot-immunogold-silver staining in the diagnosis of clonorchiasis.

All 35 confirmed clonorchiasis cases showed a positive reaction in dot-immunogold-silver staining (Dot-IGSS) with a mean serum titre of 1:1656, while none of the sera from 35 normal individuals reacted. A seroepidemiological survey of middle-school students revealed a positive rate of 17.0% (142/836), and 76.1% (105/138) of the serologically positive students were egg positive in stool examination. The egg positive rates in those with antibody at levels of 1+ to 4+ were 57.1% (28/49), 75.7% (28/37), 94.7% (36/38) and 92.9% (13/14) respectively. It is believed that Dot-IGSS can be used for the clinical diagnosis and epidemiological survey of clonorchiasis.

China↗
PubMed1976

Inclusion of satellites in an 18/21 translocation chromosome shown by ammonical-silver staining (sat-banding) in case of partial trisomy 18.

A male infant with a partial trisomy 18 and a 46,XY, --21, t(18;21)(18qter replaced by 18q12::21 p13 replaced by 21 qter) chromosome complement is described. The translocation chromosome is of special interest because it includes the satellites of chromosome 21. This was shown by differential satellite staining with the ammoniacal-silver technique.

Ammonia↗
PubMed1996

A one-day double-labelling technique for tissue specimens: immunogold-silver staining for in situ hybridization combined with alkaline phosphatase-anti-alkaline phosphatase (APAAP) immunohistochemistry for antigens.

An improved technique is described that addresses the problems of sensitivity, specificity, the use of hazardous radioactive equipment and time consumption in immunohistochemical labelling and double labelling of in situ hybridization of tissue specimens. It consists of a two-step protocol in which digoxigenin-uridine triphosphate (UTP) labelled riboprobes in the in situ hybridization step are visualized by the immunogold-silver staining method, and double labelling of tissue antigens is achieved by the application of an alkaline phosphatase-anti-alkaline phosphatase staining step. We tested this protocol using snap-frozen tissue sections of synovial tissue from patients with rheumatoid arthritis. The target mRNA was detected by perforin or cathepsin D riboprobes, the double labelling was performed using anti-collagen type IV and alpha-smooth muscle actin antibodies. It is concluded that, in comparison with an established three- to four-day double-labelling protocol used in many laboratories, this one-day combination is currently the most rapid assay of reliable quality for double labelling of in situ hybridization products and tissue antigens.

Alkaline Phosphatase↗
PubMedDate not supplied

Microwave-aided technique to detect bromodeoxyuridine in S-phase cells using immunogold-silver staining and plastic-embedded sections.

A technique is described to detect bromodeoxyuridine (BrdU) incorporated by cells in S-phase, with a monoclonal antibody, using removable plastic embedding and immunogold-silver staining (IGSS). The incubation times were reduced and the immunological reactions enhanced by microwave irradiation. The embedding in methyl methacrylate enabled us to make thinner sections and it improved the quality of the preparations. The methyl methacrylate did not hinder the reaction of BrdU with the antibody because it could be removed prior to the IGSS procedure. The IGSS procedure appeared to be very sensitive, requiring lower concentrations of the antibodies than other methods. The use of microwave irradiation shortened the time needed to stain a section from 7 to less than 4 h. Furthermore, using microwave irradiation, the concentration of the antibodies needed could be reduced even further compared with the normal IGSS procedure. In sections of the mouse testis and small intestine only nuclei of cells known to be able to proliferate appeared BrdU positive. The non-specific background staining was found to be negligible. In testes of mice that received both 3H-thymidine and BrdU more than 95% of the radioactively labelled cells also showed BrdU label and vice versa. This indicates that both methods are equally sensitive for detecting cells in S-phase.

Animals↗
PubMed1998

Differential silver-staining sodium dodecyl sulfate-polyacrylamide gel electrophoresis: a nonisotopic method for characterizing gel-separated histone-DNA complexes.

Some nonspecific, DNA-binding proteins, like the linker histones, precipitate DNA upon binding. This is a poorly understood process that limits analysis of such nucleoprotein complexes using standard gel electrophoresis. To circumvent this problem, low concentrations of glutaraldehyde were used to crosslink the linker histones to DNA; then the partially crosslinked complexes were solubilized in SDS2 and separated by SDS-PAGE. Differential detection was accomplished using two different silver staining protocols that preferentially stained either proteins or nucleic acids. A technique was developed which allows the relative proportion of linker histones and DNAs in each detected band to be determined, and is referred to as differential staining SDS-PAGE (DS-SDS-PAGE). DS-SDS-PAGE provides a novel, non-isotopic means for characterizing multiple nucleoprotein bands separated by polyacrylamide gel electrophoresis. In applying this method to a model linker histone-DNA study, we were able to detect both protein-DNA and protein-protein contacts that are important in linker histone assembly onto DNA.

Animals↗
PubMedDate not supplied

[Silver staining of the nucleolus organizer regions in the interphase cells of an embryonic pig kidney culture during and after administration actinomycin D].

Silver staining of nucleolus organizer regions was used for studying their structural alterations during inhibition of ribosomal RNA synthesis by actinomycin D and after its removal in pig kidney cells. Three nuclear types were revealed from the pattern of restored silver grains distribution in the nuclei. When the inhibitor was removed, nuclear multitype gradually disappeared for 24 hours and nuclear RNA synthesis became normal.

Animals↗
PubMed1987

[Characteristics of the silver-stained nucleoli of tumor cells from the pleural fluid of lung cancer patients].

Tumor cell nucleoli obtained from pleural effusions of 26 patients with different morphologic types of lung cancer were evaluated by silver staining. Distinct heterogeneity of tumor cell populations, with regard to the number of nucleoli as well as their functional activity in respect to ribosomal RNA synthesis, were shown to be the most common feature of all the tumors studied, regardless of their morphologic variants. One likely cause of heterogeneity in Ag nucleolar organized region (NOR) pattern of tumor cells may be due to chromosomal losses and gains from the karyotypes of acrocentric chromosomes with active NORs. Another possible cause for heterogeneity in nucleolar activity might be due to different reactions of tumor cells towards some humoral and cellular factors of pleural fluid including T-lymphocytes.

Adult↗
PubMed1989

Electrophoretic determination of ricin using immunogold silver staining--comparison with simple "protein dot" method.

Studies were performed to establish a sensitive electrophoretic immunodetection system for the highly toxic plant protein, ricin. This has potential criminal application as an agent for causing a delayed death following parenteral administration. The immunodetection system could be used to demonstrate residual traces of the toxin left in certain tissues of the victim's body. Following polyacrylamide gel electrophoresis of ricin added to rat muscle tissue extracts, the gels were electro-blotted onto nitrocellulose paper and ricin bands probed for visualisation by immunostaining. Several immunostaining procedures were investigated in order to select the most sensitive. These included indirect immunoperoxidase, peroxidase-anti-peroxidase (PAP), avidin-biotin complex (ABC) and the immunogold silver staining (IGSS) procedures. The sensitivity of PAP and indirect immunoperoxidase methods were similar at around 50 ng while the ABC technique gave visible staining of 10 ng of electro-blotted ricin. The method with greatest sensitivity was undoubtedly IGSS, which resulted in unequivocal demonstration of 4 ng of ricin. The IGSS-immunoblotting system was considered to readily demonstrate the presence of ricin in muscle tissue from the injection site of dead victims. We compared this system with the very simple method of sample dot staining. Here, samples of ricin were spotted directly onto nitrocellulose. The dots were stained using the IGSS method which was found able to demonstrate less than 10 pg of ricin.

Animals↗
PubMed2000

The use of silver-stained "comets" to visualize DNA damage and repair in normal and Xeroderma pigmentosum fibroblasts after exposure to simulated solar radiation.

The alkaline and neutral comet assays have been widely used to assess DNA damage and repair in individual cells after in vivo or in vitro exposure to chemical or physical genotoxins. Cells processed under neutral conditions generate comets primarily from DNA double strand breaks, whereas under alkaline conditions, comets arise from DNA single and double strand breaks and alkali-labile lesions. A modified version of the alkaline comet assay, as described here, used silver stain to visualize the comets and a Gelbond base to facilitate the manipulation and processing of samples. To demonstrate how these modifications improve the assay, fibroblasts derived from both normal and Xeroderma pigmentosum (Xp) individuals were exposed to simulated solar radiation and the resulting DNA damage and repair evaluated and compared with results from the relevant literature. Comets from normal fibroblasts reached their maximum length at about an hour after irradiation. Dose-dependent increases in comet length were observed up to at least 360 mJ/cm2. In contrast, comet lengths from repair deficient Xp fibroblasts were shorter than normal cells reflecting their reduced capacity to generate single strand breaks by the excision of DNA dimers. For incubation times of more than 1 h, comet lengths from normal fibroblasts underwent a time-dependent decrease, supporting the contention that this change was related to the ligation step in the DNA repair process. These changes were compatible with the model of DNA damage and repair established by others for ultraviolet radiation.

Cell Line↗
PubMed1988

Double labeling of SIgG+ cells harboring a lymphotropic herpesvirus by avidin-biotin complex immunoperoxidase and immunogold-silver staining techniques.

By reversing the usual order of double-staining procedures, we obtained optimal conditions for simultaneous detection of guinea pig lymphotropic herpesvirus (GPHLV) and surface immunoglobulin G-positive (SIgG+) cells in paraffin-embedded tissue sections. Of the different fixatives tested, Bouin's solution gave the best preservation of morphology and cell surface IgG. Double immunolabeling was best achieved when avidin-biotin complex immunoperoxidase (ABC-IP) staining was performed first for detection of intracellular viral antigen, followed by immunogold-silver staining (IGSS) for localization of cell surface IgG.

Animals↗
PubMed1990

A high resolution two-dimensional gel electrophoresis and silver staining protocol demonstrated with nuclear matrix proteins.

An improved two-dimensional gel electrophoresis procedure has been developed utilizing isolated nuclear matrix proteins. The proteins of the cellular nuclear matrix are tissue specific. They are an example of a protein set whose two-dimensional electrophoretic patterns afford much information of clinical significance. However, current two-dimensional gel techniques were not completely satisfactory for the small amounts of protein present in tissue samples. There was a need for a two-dimensional gel procedure which was capable of increased sensitivity and resolution and at the same time was reliable and reproducible. This has been accomplished by implementing several modifications to the current two-dimensional gel procedures. In addition, changes were introduced in the silver staining process of the gels to increase the signal to background ratio. The overall procedure affects a dramatic increase in the resolution and clarity of the proteins visualized on two-dimensional gels and is no more laborious than current techniques.

Antigens, Nuclear↗
PubMed1996

Demonstration of silver-stained nucleolar organizer region associated proteins (AgNORs) after wet autoclave pretreatment in breast carcinoma: correlation to tumor stage and long-term survival.

Argyrophilic nucleolar organizer region associated proteins (AgNORs) are known to reflect cellular and nucleolar activity. Due to a novel staining procedure, which substantially improves visualisation of AgNORs on formalin-fixed and paraffin-embedded material, AgNORs can be reliably demonstrated as true substructures of the nucleoli. The aim of the present study was to apply a standardized morphometric AgNOR quantification on a large series of breast carcinomas with regard to its prognostic relevance. AgNOR quantity was evaluated on archival tumor tissues of 115 adenocarcinomas of the breast treated with the wet autoclave method prior to standardized silver-staining and morphometric analysis. AgNOR parameters were correlated to prognostic features (steroid hormonal receptor status, tumor type, tumor size, histological grading, pTNM, and UICC stage) carrying out both univariate and multivariate survival analyses. AgNOR number and area were proven to be statistically significantly related (Pearson correlation coefficient: 0.67, Bonferroni adjusted P = 0.0001). Almost all AgNOR parameters, in particular CV (coefficient of variation) of corrected area (delta-area) and CV of number, were statistically significantly correlated to estrogen and progesterone receptor status as well as histological grading of tumors. Increased AgNOR parameters were statistically significantly associated with early tumor relapse and cancer related death. Univariate and multivariate analysis by means of Cox regression revealed independent prognostic significance for CV of delta-area and number of AgNORs. Various AgNOR parameters (CV of number, CV of delta-area, CV of area, mean delta-area, and mean area of AgNORs per nucleus) determined on wet autoclave pre-treated formalin-fixed and paraffin-embedded breast cancer tissues are statistically highly significantly associated with the prognostic outcome, independently predicting tumor-free and overall survival.

Adenocarcinoma↗
PubMedDate not supplied

Gel electrophoresis of giant proteins: solubilization and silver-staining of titin and nebulin from single muscle fiber segments.

Giant proteins in the megadalton range (> 0.5 MDa) appear to play important structural and functional roles in striated muscle. Titin (approximately 3 MDa) is involved in the generation of resting tension and the assembly and stability of the sarcomere in skeletal and cardiac muscle tissues, while nebulin (approximately 0.7 MDa) is thought to regulate thin filament length in skeletal muscle. Sodium dodecyl sulfate (SDS)-gel electrophoresis is an important tool in revealing the size, quantity and integrity of these giant proteins in muscle tissues. We report here a method for solubilizing, detecting and quantifying titin and nebulin from short segments of single fibers of the rabbit psoas muscle. Muscle proteins ranging from 15 kDa to 3 MDa were resolved on 3.3-12% gradient polyacrylamide gels that were silver-stained and quantitated by densitometry. Presoaking fiber segments in a low ionic strength pH 8.4 buffer enhances the amount of solubilized titin and nebulin. Solubilizing the presoaked fiber segments with SDS at 60 degrees C for 60 s maximizes the amount of intact titin; solubilizing at higher temperatures causes extensive degradation of titin. Detection sensitivity is sufficient to study titin and nebulin in fiber segments as short as 120 microns.

Animals↗
PubMed1988

An investigation of optimal gold particle size for immunohistological immunogold and immunogold-silver staining to be viewed by polarized incident light (EPI polarization) microscopy.

We investigated the optimal gold particle size for use with polarized incident light (epi polarization) microscopy with immunogold immunohistological preparation in both immunogold indirect (IGS) and silver-enhanced immunogold-silver staining (IGSS) techniques. A range of gold particle sizes from 5 nm-40 nm was used along with tissue of known immunoreactivity with a well-characterized primary monoclonal antibody. Checkerboard titrations were carried out for each technique and for each particle size. The preparations were viewed using a standard polarized incident light microscope and assessed in a semi-quantitative manner. Adequate visualization of gold particles was achieved using the indirect staining method only with a particle size of 40 nm. With silver enhancement (IGSS), particles of all sizes were clearly seen. However, 5-nm particles were considered optimal for this method because of reduced background staining, high titration of antisera possible, and crisp localization of the visual signal.

Gold↗
PubMed1992

Quality assurance and reproducibility of high-resolution two-dimensional electrophoresis and silver staining in polyacrylamide gels.

Qualitative data from high-resolution two-dimensional electrophoresis (2DE) have become clinically useful in immunoglobulin clonality analysis, in resolution of ambiguities in immunofixation typing of paraproteins, and in genetic typing of serum proteins. Since 1986, the authors have been evaluating the College of American Pathologists Reference Preparation for Serum Proteins (RPSP) as a quality control material for 2DE because (1) it is prepared exclusively from pooled human sera, (2) the pool yields a reference pattern of mixed heterozygosity for genetic markers, and (3) RPSP is widely available as a lyophilized preparation that currently serves in the authors' laboratory as a qualitative quality control preparation and that may become a quantitative quality control material or external quality assessment material for 2DE. Using the ISO-DALT 2DE system and silver-staining, the peptide patterns were examined in 11 lots of RPSP and compared with fresh serum and with each other. Consistent differences in the 2DE pattern between RPSP and fresh serum included the presence of freeze-thaw peptides, the presence of degradation spots of apolipoprotein A-I, and the diminution of apolipoprotein spot intensities in RPSP. All lots of RPSP yielded clear identification of the eight serum proteins used for quality control calculations. Run-to-run coefficients of variation for a single lot of RPSP for four parameters of 2DE spot location and gradient reproducibility were comparable with band location reproducibility for the one-dimensional procedures of serum protein electrophoresis and lactate dehydrogenase isoenzyme electrophoresis. It is concluded that the reproducibility, that is, imprecision, of 2DE is the same as one-dimensional clinical electrophoresis techniques and that either RPSP or pooled fresh serum can serve as a satisfactory internal quality control material.

Blood Protein Electrophoresis↗