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A rapid modification of the methenamine silver stain for Pneumocystis carinii and fungi employs heat alone to accelerate the silver reduction step and is suitable for both sections and smears.
With the aid of ultrasonography, representative percutaneous biopsy specimens were obtained from 20 of 21 patients (95 percent) with liver metastases of carcinoids and endocrine pancreatic tumors. The specimens were examined with silver stains and immunocytochemically after the application of monoclonal serotonin antibodies. The Grimelius argyrophil silver nitrate stain was positive in all tumor metastases, demonstrating that they were of neurohormonal endocrine type. The argentaffin reaction stained 14 of 15 metastases of small intestinal carcinoids, whereas tumors with other primary sites were unreactive. Immunocytochemical analysis with monoclonal serotonin antibodies stained all metastases of small intestinal carcinoids, and the other endocrine tumor metastases were unreactive. With immunocytochemical analysis, optimal results were obtained in Bouin-fixed tumor specimens, whereas for the argentaffin reaction, formalin was preferable. The results show that silver stains and immunocytochemical analysis with monoclonal serotonin antibodies on small percutaneous biopsy specimens of liver metastases of endocrine tumors and carcinoids are valid for the prediction of the location of the primary tumors.
Relative abundance and solubility of proteins from etiolated coleoptiles of maize were investigated using two-dimensional electrophoresis (2-D PAGE). Automatic quantification of silver-stained polypeptides on replicate 2-D gels made it possible to test the linearity of the relationship between spot integrated optical density and protein amount in the range from 15 micrograms to 135 micrograms per gel, in two inbred lines. A linear response was found for more than 60% of the spots in each genotype. When a linear response was found in both lines for a given spot, the slope values were similar in 94% of the cases, indicating the reliability of silver staining for polypeptide quantification. The parameters of the curves allowed the definition of protein classes of different abundances that could be compared for genetic variability between the two lines. From a comparison between standard 2-D patterns (trichloroacetic acid-acetone extracted proteins) and patterns obtained from Tris buffer extracted proteins, it appeared that 92% of the proteins visualized in the standard gels were soluble. No difference in genetic variability, either qualitative or quantitative, was evidenced between the various classes of abundance, or between soluble and insoluble proteins.
The cDNA fragments amplified through nested RT-PCR from the small hydrophobic (SH) protein gene and its flanking region of the five wild mumps viruses and Enders strain were subjected to single-strand conformation polymorphism (SSCP) analysis by using silver staining. These five wild isolates could be divided into three SSCP patterns and they were differentiated from the Enders strain. Two wild strains which have 3.4% heterology of the nucleotide acid sequence could be distinguished by the method. The results were well correlated with sequence analysis of the SH gene segments, indicating high applicability of the silver staining SSCP analysis for differentiation of mumps virus strains. Furthermore, the method, which is not only quick, sensitive, safe and reliable, but also reproducible and inexpensive, can be used in the molecular epidemiology study of mumps viruses.
Using the two-dimensional gel electrophoresis and two systems for computer analysis of the resulting maps, we obtained a good quantitative reproducibility of individual spots of silver-stained and Coomassie Blue-stained gels from mouse fetus liver samples. The GelScan-XL system was established for the evaluation of silver-stained gels. The Gel-Image software has shown to be capable of the analysis of Coomassie Blue-stained gels and autoradiographs. For these systems the linear relationship between defined concentrations of protein loaded on the gel and the resulting integrated intensities of spots were examined. The results show that both systems are efficient to detect quantitative changes in protein expression correlated with malformations in mouse fetuses.
Quantification of silver-stained nucleolar organiser regions (AgNORs) in paraffin sections may provide clues about the proliferation and differentiation in normal and neoplastic tissues. The aim of the present investigation was to determine whether AgNOR quantification could provide useful data about proliferation in the different segments of the normal human colorectal crypt. Samples of histologically 'normal' large intestine (n = 8) were obtained from colorectal cancer resections at a distance of > 5 cm from the tumour margins and were routinely processed for paraffin embedding using strictly standardised procedures. Sections were cut and stained with a one-stage silver colloid impregnation technique. The longitudinally sectioned crypts were divided into proliferative (P), intermediate (I) and surface (S) segments using strict criteria. Clearly defined AgNORs, which appeared as black dots within the nuclear profile, were quantified from each segment for volume density (Vv) and number per unit area (NA) estimates using traditional point-counting techniques. A 1-way analysis of variance followed by Scheffe's test indicated significant progressive reductions of AgNOR Vv and NA from P to S segments. Our data suggest that both volume and frequency of AgNORs may be related to cellular proliferation since both parameters are highest in the P segment. The further exploitation of stereological tools in conjunction with AgNOR staining may be valuable in assessing normal differentiation and proliferation patterns and in predicting the biological behaviour of neoplastic tissues in which increased proliferation is a feature.
Measurement of telomerase activity in clinically obtained tumor samples may provide important information for use as both a diagnostic marker and a prognostic indicator for patient outcome. In order to evaluate telomerase activity in tumor tissue without radiolabeling the product, we developed a simple telomeric repeat amplification protocol-silver-staining assay that is less time-consuming, is safe and requires minimal equipment. In addition, we determined the sensitivity of the silver-staining method by using extracts of telomerase-positive thyroid carcinoma cell lines which were serially diluted from 5,000 to 10 cells. Telomerase activity was also assayed in 19 thyroid tumors, 2 normal controls and 27 bone marrow aspirates. The results indicate that the technique permits the detection of telomerase activity from 5000 to as few as 10 cells. We propose that it could be immediately applicable in many laboratories due to the minimal amount of equipment required.
The intensity of the silver staining of nucleolus organizing regions (NORs) and the frequency of chromosomal associations were studied before and after antithyroid treatment in seven and eight hyperthyroid patients, respectively. For one patient, the NOR staining was significantly more intense after treatment, while for another the opposite result was found. A higher stainability of chromosome 22 was observed after treatment in six of seven patients. The difference was significant for the group of patients considered as a whole but only for one patient considered separately. Chromosome 22 also showed an increased association frequency. Fro one patient, the number of large associations and the total number of associating chromosomes was also significantly increased after treatment. A decrease of associating chromosomes 14 and 21 such as was reported by Nilsson et al, (1975) after antithyroid treatment could not be found in our sample. The possible reason for this difference are discussed.
Prostatic endocrine-paracrine (PEP) cells from the prostates of 25 radical cystectomy specimens were studied using serotonin and neuron-specific enolase immunocytochemistry and argyrophil and argentaffin silver stains. Three populations of PEP cells were identified as follows: (1) serotonin-positive only, (2) serotonin-positive and argyrophil-positive (the largest population), and (3) serotonin-positive, argyrophil-positive, and argentaffin-positive. Neuron-specific enolase immunoreactivity correlated closely with serotonin immunoreactivity. The entire PEP cell cytoplasm was serotonin and neuron-specific enolase immunoreactive, while the silver stains only stained the granulated cytoplasm. The PEP cells were present in all areas of all prostates with a surprisingly large number in the large periurethral ducts with somewhat fewer PEP cells in the prostatic urethra and smaller ducts and ductules. The peripheral acini generally contained the smallest number of PEP cells. Prostatic endocrine-paracrine cells were of the open (luminal extension), closed, and dendritic types.
In order to diagnose lung cancer at gene level, bronchial biopsy specimens from lung cancer patients, who were diagnosed by pathologic method using biopsy specimens from the same site, were used for detection of p53 gene mutation by polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP)-silver staining method. 15 of 25 (60%) specimens were found to be positive. In 10 small cell lung cancer specimens, 7 were positive; in 15 non-small lung cancer specimens, 8 were positive. These results coincided with those of other reports using surgical specimens and sequencing method. Clinical analysis showed no correlation between SSCP positivity and the patients' clinical data such as age, sex, smoking habit, stage of tumor at the time of diagnosis. It is concluded that a small piece of bronchial biopsy specimen could be used to detect p53 gene mutation instead of surgical specimens and this method might be used as an adjunct to cancer screening or for a gene diagnosis prior to gene therapy. In comparison with the routine radionuclide labelled method, silver staining method has the advantage of being simple, quick, economic, safe and convenient for clinical use.
Human lactate dehydrogenase (LDH)-A mutant gene was analyzed by polymerase chain reaction - DNA conformation polymorphism (DCP). We used polyacrylamide gradient gel and silver staining procedures for DCP analysis and observed abnormal migration patterns in individuals heterozygous for LDH-A deficiency. Further sequence determination of the mutant alleles consistently resulted in detection of base substitutions, a G to T transversion at codon 328 (GAG----TAG), and synonymous substitutions at codon 115, 160 and 172. Such mutations were easily detectable using the DCP technique. The DCP technique using the polyacrylamide gradient gel and silver staining method seems likely to be useful for the rapid screening of mutations and for further genotype detection.
The silver-staining technique was used to study several cases of translocations involving acrocentric chromosomes: robertsonian translocations, balanced reciprocal translocations, non-reciprocal translocation. This technique provides very useful and reliable information about the presence, loss or translocation of active NORs in cases of acrocentric breaks.
In situ hybridization using biotinylated DNA probes has become an important tool in histopathology. It is well known that the sensitivity of the methods used to demonstrate viral DNA in formalin-fixed and paraffin-embedded specimen depends strongly on the detection system used. In the present study, an optimized in situ DNA hybridization protocol was combined with four different approaches of gold-silver staining methods. For silver enhancement, the recently described method of silver acetate autometallography, a technique allowing highly efficient development without the necessity of dark room illumination has been used. The most efficient detection method found in our experiments was the use of gold-adsorbed anti-biotin antibodies with subsequent silver enhancement. This staining procedure can be completed in 5 hours including hybridization and is a highly sensitive alternative to peroxidase and alkaline phosphatase detection systems.
A silver staining technique to be used on isoelectric focusing (IEF) gels for cerebrospinal fluid (CSF) oligoclonal IgG bands is described. The technique provides a sensitivity for detection of greater than 0.17 micrograms of IgG. The additional use of immunofixation with anti-human IgG antiserum rather than chemical fixation improves the sensitivity two-fold and permits identification of the oligoclonal bands as IgG. The technique enables one to focus small volumes of unconcentrated CSF (less than 40 microliters) and provides a powerful research tool.
A modification of the silver staining procedure of Howell and Black (1980) is reported which makes use of teleostean gelatin as protective colloid and renders a high signal-to-noise ratio. We demonstrate that this ratio can be further increased by subsequent attenuation with a chemical reducer consisting of a mixture of potassium ferricyanide (III) and sodium thiosulphate. It is shown that slight changes of the concentration of the reactive compounds of the chemical reducer make the protocol applicable to human, plant (Aliium cepa, Rhinanthus minor) as well as meiotic insect (Acheta domesticus) chromosome preparations. Due to its broad applicability, the method could find utilization in studies on chromatin and chromosome functions in many species.
Silver staining of nucleoli reveals argyrophilic proteins associated with nucleolar organizer region (Ag-NOR) proteins. Argyrophilic components appear as dots about 1 micron in diameter dispersed throughout the nucleolus (Ag-NOR dots). The count of Ag-NOR dots is a useful index for improving the cancer diagnosis and determination of prognosis. Here we describe software developed on a medium-cost image analyzer in order to evaluate the mean area of NORs and their number relative to an internal reference, the number and areas of clusters of NORs and the area of the nucleus. Statistical analysis of the data was performed during counting. The first application concerned counting NOR dots during mitosis in cell imprints; those counts were 2.3, 15.3 and 55.56 for the metaphase, telophase and interphase, respectively (relative to unitary dots of metaphase cells). In the second application we demonstrated a significant difference in NOR numbers between two groups of prostatic cancers with good and poor prognoses (6.05 +/- 2.79 SD and 7.96 +/- 3.01, respectively; with Student's t test, = 1.999; P = .05).
BACKGROUND: Viral hepatitis due to hepatitis B virus and hepatitis C virus are major public health problems all over the world. Traditional detection methods including polymerase chain reaction (PCR)-based assays and enzyme-linked immunosorbent assays (ELISA) are expensive and time-consuming. In our assay, a protein chip assay using Nano-gold Immunological Amplification and Silver Staining (NIASS) method was applied to detect HBV and HCV antibodies rapidly and simultaneously. METHODS: Chemically modified glass slides were used as solid supports (named chip), on which several antigens, including HBsAg, HBeAg, HBcAg and HCVAg (a mixture of NS3, NS5 and core antigens) were immobilized respectively. Colloidal nano-gold labelled staphylococcal protein A (SPA) was used as an indicator and immunogold silver staining enhancement technique was applied to amplify the detection signals, producing black image on array spots, which were visible with naked eyes. To determine the detection limit of the protein chip assay, a set of model arrays in which human IgG was spotted were structured and the model arrays were incubated with different concentrations of anti-IgG. A total of 305 serum samples previously characterized with commercial ELISA were divided into 4 groups and tested in this assay. RESULTS: We prepared mono-dispersed, spherical nano-gold particles with an average diameter of 15 +/- 2 nm. Colloidal nano-gold-SPA particles observed by TEM were well-distributed, maintaining uniform and stable. The optimum silver enhancement time ranged from 8 to 12 minutes. In our assay, the protein chips could detect serum antibodies against HBsAg, HBeAg, HBcAg and HCVAg with the absence of the cross reaction. In the model arrays, the anti-IgG as low as 3 ng/ml could be detected. The data for comparing the protein chip assay with ELISA indicated that no distinct difference (P > 0.05) existed between the results determined by our assay and ELISA respectively. CONCLUSION: Results showed that our assay can be applied with serology for the detection of HBV and HCV antibodies rapidly and simultaneously in clinical detection.
We have developed a reliable method for silver staining nodules on synaptonemal complexes (SCs) of tomato (Lycopersicon esculentum). This technique involves hypotonically bursting primary microsporocytes, fixing SC spreads with paraformaldehyde, and incubating the spreads at 40 degrees C in a 33% aqueous silver nitrate solution covered with nylon mesh. When tomato SCs were stained by this method, nodules were observed with the same distribution and frequency as nodules stained with uranyl acetate and lead citrate. Incubation in silver nitrate at higher temperatures caused the loss of some or all nodules. The pattern of loss suggests that two types of nodules coexist during late zygonema and early pachynema and that one type becomes the late nodules of mid-pachynema through early diplonema.