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PubMed1995

Comparison of silver-staining nucleolar organizer region (AgNOR) counts and proliferating cell nuclear antigen (PCNA) expression in reactive mesothelial hyperplasia and malignant mesothelioma.

Previous workers have used counts of colloidal silver-stained nucleolar organizer regions (AgNORs) to help distinguish reactive from malignant mesothelial processes. We sought to compare the demanding technique of AgNOR counting with the identification of proliferating cell nuclear antigen (PCNA) in 10 pleural malignant mesotheliomas and 11 reactive mesothelial proliferations. The mean AgNOR count was significantly higher in malignant compared to reactive lesions (5.10 [95% confidence interval, CI 4.35-5.84] vs 3.68 [95% CI 3.17-4.19], p = 0.004), as was the PCNA index (26.9 [95% CI 17.37-36.49] vs 9.67 [95% CI 4.94-14.39], p = 0.004). Less overlap was seen with PCNA indices and no reactive mesothelial proliferations had a PCNA index greater than 20%, suggesting that scores over this level may be a specific indicator of malignancy in this setting. There was only a weak correlation between the 2 proliferation indices suggesting that PCNA and AgNORs are present at somewhat different times of the cell cycle and/or persist in the post-S phase cell for variable lengths of time.

Adult↗
PubMed2003

Identification of nuclear proteins of small cell lung cancer cell line H82: An improved procedure for the analysis of silver-stained proteins.

An efficient method for digestion and extraction of proteolytic peptides from silver-stained proteins was applied to the characterization of nuclear proteins from the small cell lung cancer H82 (ATCC HTB 175) cell line previously separated by high-resolution large format two-dimensional gel electrophoresis. From 68 spots, evenly distributed on the gel area and representing a wide range of spot intensities, 63 (92%) were successfully identified by matrix-assisted laser desorption/ionization (MALDI) or electrospray ionozation-mass spectrometry (ESI-MS). In five cases where the identification was not possible, the presence of an intense background apparently due to the leakage of polymers from the microtubes or other plastics, was detected. Extensive analysis of peptide sequences by ESI MS/MS experiments allowed the identification of post-translational modifications, such as acetylation, phosphorylation, deamidation of asparagine residues and the presence of isoaspartic acid. A new protein variant not reported in sequence databases was also detected.

Amino Acid Sequence↗
PubMed1985

Silver staining of meiotic chromosomes in the fungus, Coprinus cinereus.

We have taken advantage of the synchronous meiotic process in the basidiomycete Coprinus cinereus to develop a simple and rapid method to selectively stain meiotic chromosomes and nucleoli in this fungus without prior removal of the cell wall. Electron microscopic examination of these silver-stained chromosomes indicated that the lateral elements of the synaptonemal complexes were prominently stained, and terminal attachment plaques were apparent. We found that a translocation quadrivalent could be recognized easily in the light microscope using these methods. The procedures appear suitable for the characterization of chromosome rearrangements in this small genome, and should facilitate cytogenetic analysis in this fungus.

Basidiomycota↗
PubMed1986

Spatial visualization of junctional complexes by backscattered electron imaging and silver staining.

A method for the observation of junctional complexes by backscattered electron imaging in scanning electron microscopy, in tissue blocks, is presented. The junctional complexes are revealed by a modified silver staining method (originally devised for nucleolar organizer regions), used "en bloc" after formalin or glutaraldehyde fixation. Backscattered electron imaging allows, after this staining, the observation of junctional complexes through the surface of intact superficial cells, in the three tissues studied (liver, jejunum and urinary bladder). The interest of this approach is to offer the possibility of observing junctional complexes "by transparence," in nondissociated and nonsectioned tissues.

Animals↗
PubMed1998

Sequential silver staining and in situ hybridization reveal a direct association between rDNA levels and the expression of homologous nucleolar organizing regions: a hypothesis for NOR structure and function.

We have developed a procedure for sequential silver staining and in situ hybridization to analyze the relationship between the amount of rDNA present in nucleolar organizer regions, as estimated by in situ hybridization, and their level of expression, as estimated by the silver signal. For simplicity we used cells from the insectivorous mole Talpa occidentalis, which have a single pair of nucleolar organizer regions in chromosome pair 3. The relative content of ribosomal cistrons was also related to the hierarchy of activation of the nucleolar organizer regions present in this chromosomal pair. Statistical analyses demonstrated that both the relative level of expression and the activation hierarchy depended mainly on the number of ribosomal cistrons in nucleolar organizer regions. We propose a functional two-step hypothesis, which is consistent with most known data concerning interchromosomal, intercellular and interindividual variation in a number of plant and animal species, including Talpa occidentalis. In step one, the first available transcription factors bind randomly to the ribosomal promoters, such that larger nucleolar organizer regions are more likely to recruit them. In the second step the remaining transcription factors are recruited in a cooperative way, thus completing activation of one nucleolar organizer region, before the next one becomes active.

Animals↗
PubMed1990

Use of enhanced silver staining combined with electron microscopical immunolabelling to demonstrate the colocalization of neuropeptide Y and vasoactive intestinal polypeptide in cerebrovascular nerves.

The combination of immunolabelling at the electron microscope level and enhanced silver staining has been used to demonstrate the colocalization of neuropeptide Y and vasoactive intestinal polypeptide in perivascular nerves supplying cerebral arteries of the rat. This has been shown in control tissue, but it is easier to demonstrate after long-term sympathectomy since that leads to an enhancement of neuropeptide Y in vasoactive intestinal polypeptide-containing parasympathetic nerves supplying these vessels. Immunolabelling of the antigens for these peptides was performed sequentially with the biotin streptavidin diaminobenzidine method, and the end product to the first antiserum was gold-silver intensified before the visualization of the second antigen. Using this technique, it was shown that all the neuropeptide Y immunoreactivity present in the rat cerebral vessels after long-term sympathectomy with guanethidine was localized in vasoactive intestinal polypeptide-containing nerves. Furthermore, an immunohistochemical analysis of the parasympathetic pterygopalatine ganglia in guanethidine-treated rats showed an increase in the percentage of neurons displaying neuropeptide Y immunoreactivity. In order to clarify if the pterygopalatine ganglion was the origin of those neuropeptide Y/vasoactive intestinal polypeptide-immunoreactive cerebrovascular nerves, which had increased in number after sympathectomy, a fluorescent neuronal tracer (Fast Blue) was applied to the right middle cerebral artery of rats which had undergone guanethidine treatment for six weeks. Immunohistochemical analysis of the ipsilateral ganglion 72 h after application of the tracer revealed the presence of immunoreactivity to both these peptides in retrogradely labelled neurons. It is concluded that neuropeptide Y and vasoactive intestinal polypeptide are colocalized in perivascular parasympathetic nerves supplying the middle cerebral artery of the rat, which have their origin in the pterygopalatine ganglion. Furthermore, long-term sympathectomy with guanethidine leads to an increase in the expression of neuropeptide Y in these vasoactive intestinal polypeptide-immunoreactive neurons.

Animals↗
PubMed1984

Use of the Bio-Rad silver stain to identify gel purified RNA components of small nuclear ribonucleoprotein antigens.

The ultrasensitive silver staining procedure developed for the detection of proteins in polyacrylamide gels has been used to identify the RNA component of small nuclear ribonucleoprotein antigens recognised by antinuclear antibodies. The technique described is a simple, safe and inexpensive procedure with a sensitivity comparable with that obtained by biosynthetic labelling of RNA with 32P.

Antibodies, Antinuclear↗
PubMed1996

Investigation of megakaryopoiesis in myelosuppressed bone marrow using immunogold-silver staining (IGSS).

To determine the frequencies and differential counts of megakaryocytes after cytoreductive treatment in nucleated low-density (1.060 g/ml) bone marrow cells (BMNC) of dogs an immunogold-silver staining (IGSS) technique with the lineage specific monoclonal antibody 2F9 was established. This antibody recognizes the glycoprotein IIb/IIIa complex expressed on the surface of canine megakaryocytes and platelets. The IGSS technique enables not only the detection of megakaryocytes occurring at a low frequency (0.1-0.2%), but also the discrimination between the different maturation stages of megakaryocytes due to cell size, nuclear morphology and cytoplasmic staining. By the use of this technique, small lymphoid megakaryocytic cells were identified. Comparable numbers of megakaryocyte colony-forming cells in 2F9-depleted and nondepleted BMNC suspensions (25.7 +/- 5.0 vs. 25.3 +/- 5.1 Meg-CFC/10(5) BMNC) indicate that these small 2F9 positive cells are nonclonogenic precursors of megakaryoblasts. To prove the applicability of IGSS, serial examinations of bone marrow samples from dogs treated with recombinant human interleukin-6 (IL-6) after exposure to 2.4 Gy total body irradiation (TBI) were performed. The results of the microscopic evaluation indicate that, in the recovery phase after TBI, IL-6 induced an earlier and stronger increase in megakaryocyte frequency in comparison to the control. Interestingly, all maturation stages of the megakaryocytic lineage took part in this IL-6 induced improvement of megakaryocyte recovery.

Animals↗
PubMed1984

Nucleolar silver-stained granules in maturing erythroid and granulocytic cells.

Human and rabbit erythroid and granulocytic precursors in bone marrow have been investigated to provide information concerning the number of nucleolar silver-stained granules (SSGs), which represent active interphasic nucleolar organizer regions (NORs). The differentiation and maturation of precursor cells of both investigated cell lines are characterized by a gradual decrease in number of nucleolar SSGs. In advanced maturation stages of erythroblasts or granulocytes, which are known to lose the capacity to divide, the number of nucleolar SSGs is smaller than the reported average or maximal values of NORs determined for human or rabbit cells. Since committed stem cells from both cell lines contain several times the number of nucleolar SSGs than the last dividing maturation and differentiation stages, the number of active parts of interphasic NORs in committed stem cells seems to be increased and might represent a stock for the later stages. In addition, the number of nucleolar SSGs appear to be a very convenient marker of nucleolar biosynthetic activity in individual differentiating and maturing blood cells. The differences between erythroid and granulocytic stem cells with respect to the number of nucleolar SSGs disappear during the course of further differentiation and maturation.

Animals↗
PubMed1994

Do silver stained structures in vaginal parabasal cells identify a different population of cells in buffaloes?

A modified single step silver colloidal staining method for interpretation of routine vaginal smears of buffaloes which aids in analysing every component under light microscope with minimum efforts has been suggested, and it can be adopted in all clinical laboratories. A novel population of parabasal cells has been described which was distinct in morphology and nuclear distribution of silver stained structures (SSS). These SSS has been designated as the 'parabasal dots'. The significance of these cells needs further investigation and confirmation.

Animals↗
PubMed1984

Silver staining of DNA restriction fragments for the rapid identification of adenovirus isolates: application during nosocomial outbreaks.

The ultrasensitive photochemical silver stain for nucleic acids, described by Beidler et al. (1982), has been applied to the detection of adenovirus restriction fragments as a relatively rapid technique for the identification of virus isolates. In this study, restriction enzyme cleavage analysis was used to characterize adenovirus isolates from what appeared to be two nosocomial outbreaks. The first outbreak was thus shown to include two clusters of patients, and involved two serotypes Ad7c and Ad40. The second outbreak was unrelated and involved Ad35. Although restriction analysis does not replace serum neutralization as a routine method for typing adenoviruses, it is a much more rapid means of discriminating between different patient isolates, providing a current rather than retrospective analysis of a nosocomial outbreak. During the first outbreak, restriction analysis identified two distinct adenovirus serotypes from one patient--Ad7c from a nasopharyngeal aspirate and Ad41 from a stool specimen. Restriction analysis is also valuable for the sub-typing of virus isolates. In this study, the Ad40 and Ad41 isolates were shown to be variants of the respective prototype strains.

Adenoviridae Infections↗
PubMed1984

"Ultrasensitive" silver stains: their use exemplified in the study of normal human cerebrospinal fluid proteins separated by two-dimensional electrophoresis.

We have developed a series of photochemical silver stains that are more than 100 times as sensitive as the Coomassie Blue stains. These stains facilitate the quantitative estimation of proteins in two-dimensional gel patterns. Recently, we discovered that by using light (photodevelopment) to catalyze the reduction of ionic silver to metallic silver, we could produce an image of proteins on polyacrylamide gels within 10 min after their electrophoretic separation. We used these stains to detect proteins resolved in two-dimensional electrophoretograms of lumbar cerebrospinal fluid from 51 normal human volunteers, finding no qualitative protein changes in the more than 300 CSF polypeptides resolved on each individual's electrophoretogram. We quantified 67 of these proteins per individual with computer-assisted densitometry, only five of which showed significant quantitative age-related alterations. We saw no sex-related differences. This consistency in the protein pattern for cerebrospinal fluid from normal individuals allows it to serve as a baseline for comparisons with disease states.

Adult↗
PubMedDate not supplied

Diagnostic value of silver-stained interphasic nucleolar organizer regions in breast tumors.

Seventy-six specimens of normal breast tissue and benign and malignant breast lesions were studied to assess the mean area occupied by silver-stained proteins of the nucleolar organizer regions (MNORA) of the nucleolus. The assessment was performed with a computer-assisted image analyzer. The results indicate that only 30% of malignant lesions have a MNORA value greater than that of normal breast tissue or benign lesions. On the other hand, MNORA values of ductal carcinoma in situ were significantly greater than those of epitheliosis (papillomatosis). MNORA values were also significantly different in grade I and grade III invasive ductal carcinomas, the latter exhibiting the highest MNORA values of all the cases observed. Evaluation of MNORA values may therefore help in differentiating benign epithelial proliferations from ductal carcinomas in situ. Furthermore, because there is evidence that MNORA values are indicative of the cell duplication rate, MNORA values may ultimately be considered an objective prognostic parameter in addition to grading for invasive ductal carcinomas.

Adenofibroma↗
PubMed1989

Simultaneous detection of allotypes in native and activated human C2 by isoelectric focusing and silver staining.

A simple and highly sensitive analytical isoelectric focusing (IEF) technique using immunofixation with anti-C2 serum followed by silver staining has been developed in order to study simultaneously the structural polymorphism of both native C2 and C2 activation fragments (C2a MW 74,000 and C2b MW 34,000). Structural C2 polymorphism of C2*B and C2*C allotypes (but not of C2*A1) was found to be associated with the C2a fragment, whereas C2b appears to display no polymorphism. Commercially available IEF gels and C2 antisera gave reproducible allotyping data and make this technique of general use for densitometric analysis of native and activated C2. In vivo C2 activation was studied through C2a/native C2 area ratios obtained by computerized densitometry. Significantly lower ratios were observed in healthy individuals than in patients with systemic lupus erythematosus (SLE), reflecting an abnormally high classical pathway activation of complement in SLE. This methodology may be of value for immunogenetic and functional studies of other complement components.

Alleles↗
PubMed1992

Immunogold silver staining method for light microscopic detection of peripheral blood lymphocyte surface antigens with monoclonal antibodies.

Labelling of peripheral blood Lymphocytes surface antigens was carried out using the method of colloidal gold, enhanced with silver staining. Instead of PBS the minimal essential medium (MEM) according the Eagle, pH 7.2, was used rinsing of isolated lymphocytes. Visibility of positive reactions on lymphocytes at application of both mentioned media was the same. Positive reaction at demonstration of p24 BLV on cells acquired the from of black cap while the IgG expression was observed in the from of diffuse dispersion of colloidal gold on cells. Differences between the application of individual media were observed in the shape of peripheral lymphocytes in smears. Utilization of Eagle's MEM resulted in more uniform shapes and optically smooth surfaces when viewed under a light microscope.

Animals↗
PubMed1978

Rapid identification of chromosomes carrying silver-stained nucleolus-organizing regions: application to case of 21/21 Robertsonian translocation.

The use of a combination of transmitted light and epiluminescence after silver and fluorescent staining of chromosome preparations makes it possible to achieve simultaneous visualization of silver-stained NORs and flourescent chromosomes. This technique permits exact localization of silver precipitates on normal and BrdU-substituted chromosomes. After previous silver impregnation, fluorescent staining by actinomycin-daunomycin-DAPI was used to induce a banding pattern that enables identification of specific chromosomes while observing silver-stained NORs at the same time. Application of this method to a Down's syndrome patient revealed a 21/21 Robertsonian translocation with NOR'S eliminated.

Cell Nucleolus↗
PubMed1989

[A quantitative study of Ag-positive nucleolar segments revealed by silver staining in the interphase nuclei of trophoblast cells from the rat placental connective zone].

A cytomorphological study was made of silver stained nucleoli in interphasic nuclei of trophoblast cells from the rat placenta connective zone, in addition to calculation of Ag-positive spherules in the nucleoli. The prevalent number of Ag-positive nucleolar spherules in the nuclei was 6, corresponding to the number of nucleolar organizers (NOR's) in the diploid chromosome complement of the rat. The mean number of Ag-positive spherules in the nucleoli progressively increase in the course of polyploidization from 2c to 32c; variability of the spherule number also increasing. The mean area of nucleoli is found to increase in proportion to the ploidy degree. A high correlation is found between the number of Ag-positive spherules and the area of nucleoli in the nucleus (r = 0.78). This appropriateness is exhibited at all the ploidy levels. The number of Ag-spherules and the area of nucleoli are found to depend slightly on the number of nucleoli. The possibility to use the number of Ag-positive spherules as a criterion of the activity of the NOR in interphasic nuclei is discussed.

Animals↗