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PubMed1987

The activity of nucleolar organizer regions of human bone marrow cells studied with silver staining. II. Acute leukemia.

The activity of nucleolar organizer regions (NOR) in chromosomes and interphase nuclei of bone marrow cells from 11 adult patients with acute lymphoblastic leukemia (ALL), 35 patients with acute nonlymphoblastic leukemia (ANLL), and eight healthy donors has been studied with silver nitrate staining. PHA-stimulated lymphocytes of the same individuals were used as standards of the maximum silver-staining patterns for each person. In 90% of patients with acute leukemia the average number of Ag+NOR in metaphases was lower when compared with that of PHA-stimulated lymphocytes. A variable expression of NOR was observed within the cell population and between individual patients. The populations tested showed high heterogeneity in relation to the content of Ag-negative mitoses. Ag+NOR per metaphase and the content of Ag-negative mitoses in bone marrow did not differ between patients with ALL and ANLL. Differences in the staining pattern in leukemic cells are discussed.

Acute Disease↗
PubMed1990

[Morpho-functional characteristics of normal and pathological human megakaryocytes studied by selective silver staining of cell nucleoli].

The activity of nucleolar organizer (NO) in megakaryocytes (MG) from 8 donors, 10 patients with immune thrombocytopenias (IT), 17 patients with chronic myelocytic leukemia (CML) and 14 patients with multiple myeloma (MM) was studied by silver staining. The average number of nucleoli in MG of normal donors comprised 21.8 per nucleus with a range from 16.6 to 33.7. It was significantly lower in MG of CML patients, and, on the contrary, it was higher in MG of IT patients. The average number of Ag grains per nucleus reflecting their activity in relation to ribosomal RNA synthesis was found to be the highest (127 +/- 32.1) in MG of IT patients but rather low (43.2 +/- 7.2) in CML patients as compared to those of the control (76.5 +/- 11.1) and MM patients (86.0 +/- 5.6). The differences in the functional state of MG in varying diseases as well as possibilities of using this new approach in hematology have been discussed.

Cell Nucleolus↗
PubMed1977

Ultrastructural distinction between reticular and collagenous fibers with an ammoniacal silver stain.

Reticular and collagenous fibers stain differently when subjected to ammoniacal silver reduction. A variety of tissues were subjected to such a "reticulin" technique and the association of reaction product with intercellular connective tissue elements was studied with the electron microscope. The reaction with reticular fibers was primarily associated with the interfibrillar matrix, and was globular in form having a wide variety of particle sizes. Conversely, in dermal collagen the unit fibrils were stained rather than the interfibrillar matrix. The precipitate was punctate in form and was associated with the cross striations of unit collagen fibrils. Large microfibrils also reacted positively with the stain, imparting a faint periodicity. Basement membranes were stained uniquely. The underlying plasmalemma and the lamina densa were heavily stained with silver while the lamina lucida was relatively unstained. The unit fibrils of the lamina reticularis stained in the same manner as dermal unit collagen while the ground substance remained unstained. This represents a clear distinction between the argentophilic characteristics of collagenous fibers, reticular fibers, and basement membranes.

Animals↗
PubMed1981

The silver-staining technique--a tool for characterizing lymphocyte populations in mammalian peripheral blood.

Nucleolus organizer regions (NORs) of the peripheral blood lymphocytes (PBL) of 9 mammalian species were analyzed by means of a silver-staining procedure. Species-specific NOR patterns were demonstrated. The number of NOR chromosomes was positively correlated with the nucleolar coefficient, and negatively correlated with the relative frequencies of uninucleolar cells in PBL interphase.

Animals↗
PubMed1998

Protein identification at the low femtomole level from silver-stained gels using a new fritless electrospray interface for liquid chromatography-microspray and nanospray mass spectrometry.

Conventional capillary liquid chromatography/mass spectrometry (LC/MS) typically employs low microl/min flow rates with gas/liquid sheath to enhance spray stability. Over the past several years a number of reports have demonstrated success with electrospray (ES) interface designs optimized for submicroliter/min flows which have clear advantages in terms of enhancement of detection limit, lower sample consumption, and ability to accommodate a wider range of buffer conditions. We report here a fritless electrospray interface (FESI) design that is inexpensive and robust and can be operated and adapted to accommodate a variety of applications for submicroliter/min flow rates. The novelty of this interface revolves around the use of a fritless microcapillary column and precolumn application of electrospray voltage at a microtee junction to achieve stable microspray and nanospray flow rates. This sheathless FESI device eliminates postcolumn dead volume since small particles (</= 10 micron) are packed directly into laser-pulled fused silica capillary needles from which a spray originates. For analysis of proteins/peptides in solution, low femtomole sensitivity has been achieved (attomoles for selected-ion monitoring), while low nanogram sensitivity was attained for proteins derived from in-gel-digested silver-stained bands from 1-D and 2-D gels. Several applications for tandem MS protein/peptide identification using LC-microspray, LC-nanospray, or infusion nanospray are presented.

Amino Acid Sequence↗
PubMed2005

A comparison of immunohistochemical and silver staining methods for the detection of diffuse plaques in the aged canine brain.

Plaques and amyloid angiopathy represent frequent findings in the aging canine brain, whereas neuritic (senile) plaques and neurofibrillary tangles, the hallmark of Alzheimer's disease in humans, have only rarely been described in this species. Most studies on canine age-related CNS changes have used the modified (mod.) Bielschowsky method. Therefore, it remains to be determined, whether results obtained represent species-specific age-related changes or are due to the use of inappropriate methods. Consequently, the aim of this study was to compare the usefulness of various methods for the detection of age-related changes in the canine CNS. In a preliminary study, 130 brains from dogs between 1 month and 18 years of age were screened for the presence of A-beta protein using a monoclonal antibody against the beta-amyloid peptide. Thereafter, 30 brains of aged dogs, 10-18 years of age, out of the 130, were comparatively investigated by immunohistochemistry and silver stains such as the method of Campbell-Switzer, Reusche, Gallyas, modified (mod.) Bielschowsky and mod. Bodian as well as the Congo red-stain. Vascular amyloid angiopathy, plaques and A-beta protein deposits in the hippocampus/dentate gyrus were an age-dependent process starting at the age of 8 years and increased in a linear fashion with age. In contrast, plaque density did not rise in a similar age-related fashion in aged dogs. Moreover, great individual variations were observed in aged animals. Immunohistology was by far the most sensitive method for detection of diffuse plaques and amyloid angiopathy, followed by the Campbell-Switzer, Reusche, mod. Bielschowsky, and mod. Bodian technique. Neurofibrillary tangles and Congo red positive neuritic plaques were not found with any method. Immunohistochemically, two plaque types, both resembling diffuse human plaques, occurring in the superficial or deep cortical layers, were detected. Cluster analysis suggested the presence of three subgroups such as aged dogs with a low, medium and high number of plaques. Summarized, detection of plaques depends strongly upon the method used and the aging canine brain displays diffuse plaques and amyloid angiopathy but lacks neurofibrillary tangles and classic/neuritic and Congo red positive plaques. Cluster analysis indicated, that genetic factors might be important as predisposition for the development of diffuse plaques in dogs. The observed findings substantiate previous observations, that the canine brain represents an important model to study the pathogenesis of diffuse plaques and amyloid angiopathy in the absence of other Alzheimer disease characteristic lesions.

Age Factors↗
PubMed2002

Improvement of ciliate identification and quantification: a new protocol for fluorescence in situ hybridization (FISH) in combination with silver stain techniques.

A new protocol for taxon specific probe based fluorescent in situ hybridization was developed for the identification and quantification of ciliates in microbial communities. Various fixatives and experimental parameters were evaluated and optimized with respect to cell permeability and morphological preservation. Optimum results were adaption by obatined of a modified fixation method using Bouin's solution. Furthermore, conventional staining procedures such as different Protargol stain techniques and a silver nitrate impregnation method were modified and can now be applied in combination with fluorescence in situ hybridization. The new protocol allows a rapid and reliable identification as well as quantification of ciliates based upon classical morphological aspects and rRNA based phylogenetic relationships performed in one experiment. Furthermore, a set of specific probes targeting different regions of the 18S rRNA was designed for Glaucoma scintillans Ehrenberg, 1830 and tested by applying this new approach of combining in situ cell hybridization with conventional staining techniques.

Animals↗
PubMed1988

Sensitive detection of immunogold-silver staining with darkfield and epi-polarization microscopy.

We evaluated the contribution of darkfield and epi-polarization microscopy to the detection of leukocyte cell surface antigens with immunogold-silver staining (IGSS). Lymphocyte cell surface differentiation antigens were labeled with monoclonal antibodies and IGSS as described for brightfield microscopy. In darkfield and epi-polarization microscopy the labeling appeared as bright spots on a dark background. The sensitivity of detection was much higher than that of brightfield microscopy. Sixteenfold higher dilutions of the monoclonal antibody could be used to detect all cells expressing the antigen in the cell suspension. However, non-specific staining was also better visualized. The latter could be reduced to a level comparable to that of brightfield microscopy only by use of weaker labeling conditions. A 25% reduction of the silver enhancement time was necessary for this purpose. However, these weaker labeling conditions also reduced the intensity of the specific staining. Therefore, the efficiency of IGSS, as detected with darkfield and epi-polarization microscopy, was only fourfold greater than that found with brightfield microscopy or that of an immunofluorescence procedure. Especially in combination with transmitted light, to improve cell identification, epi-polarization microscopy is a reliable and sensitive method for detection of immunogold-silver-labeled cell surface antigens for diagnostic and research purposes.

Antibodies, Monoclonal↗
PubMed1989

Detection of oligoclonal IgG bands in unconcentrated CSF in multiple sclerosis and other neurological diseases by isoelectric focusing on ultrathin-layer polyacrylamide gel immunofixation and silver staining.

Isoelectric focusing of proteins (IEF) in ultrathin-layer polyacrylamide gel (0.4 mm, PAG), followed by direct immunofixation with monospecific antiserum and silver staining, is a highly specific, sensitive and simple method for the demonstration of oligoclonal IgG in unconcentrated cerebrospinal fluid (CSF) samples (5-10 microliters). For the present method, the optimal concentrations of IgG in CSF samples are about 0.025-0.030 g/l, corresponding to the applied amount of 125-150 mg. In our testing of this method, oligoclonal IgG bands in CSF specimens were clearly demonstrated in 52 (96%) of 54 patients with clinically established definite diagnosis of multiple sclerosis (MS), in 4 (40%) of 10 patients with infectious diseases of the CNS, and in 9 patients (25%) of 38 with other neurological diseases. Abnormal patterns were also demonstrated in the serum of patients with MS (43%). Intrathecally synthesized IgG was mathematically calculated in 43 (80%) out of 54 patients with MS. This method appears to be a useful alternative for the demonstration of oligoclonal IgG bands in the unconcentrated CSF, especially when questionable or negative results arise by routine electrophoretic technique for oligoclonal bands detection.

Humans↗
PubMed1993

Immunogold-silver staining and epipolarized light microscopic detection of phosphoenolpyruvate carboxykinase and glycogen phosphorylase in rat liver.

The subcellular distribution of enzymes related to carbohydrate metabolism was determined in sections of paraformaldehyde fixed and polyethylene glycol-1540-embedded rat liver and in cryostat sections. For this purpose, goat anti-rat phosphoenolpyruvate carboxykinase (PEPCK) serum and rabbit anti-rat glycogen phosphorylase (GP) serum were used as primary antibodies to localize the corresponding antigens. The primary antibodies were localized by 5 nm colloidal gold labeled secondary antibodies (either rabbit anti-goat IgG for PEPCK or goat anti-rabbit IgG for GP), and the gold particles were enhanced by silver staining using appropriate development reagents. The silver enhanced gold particles were detected by epipolarized light microscopy. PEPCK and GP immunoreactive molecules were found only in glycogen-containing areas of the cytosome of hepatocytes, and not in other cells. No immunocytochemical staining of hepatocytes was found when normal serum replaced the primary antibody in the procedures. Visio-Bond semithin (0.35-1.0 micron) sections provided higher resolution for subcellular immunostaining of PEPCK and GP than cryosections of 10 microns. Epipolarized light microscopy provided detection at high sensitivity of the gold-labeled antibody, and combined with transmitted light, allowed simultaneous visualization of the tissue morphology.

Animals↗
PubMed1992

Changes of silver-stained nucleolar organizer regions in mouse endometrial carcinogenesis induced by N-methyl-N-nitrosourea and 17 beta-oestradiol.

A high incidence of endometrial adenocarcinoma and pre-neoplastic lesions was induced in ICR mice treated with N-methyl-N-nitrosourea and 17 beta-oestradiol within 23 weeks. The endometrial lesions were histopathologically similar to those of human subjects. To assess the cell proliferative activity of these lesions, a one-step silver colloid staining for nucleolar organizer regions was applied and the numbers of silver-stained nucleolar organizer regions (AgNORs) were counted. The mean numbers +/- SD of AgNORs in each lesion were as follows: simple hyperplasia, 2.07 +/- 0.36; complex hyperplasia without cytological atypia, 2.79 +/- 0.39; complex hyperplasia with cytological atypia, 3.43 +/- 0.38; and well-differentiated adenocarcinoma, 4.17 +/- 0.40. Significant differences were observed in each lesion (P < 0.001). These findings suggest that the mean numbers of Ag-NORs are increased in the progression of neoplastic changes in the mouse endometrium, as in human endometrial lesions. This rapid induction model of endometrial carcinoma in mice is useful in the understanding of the histogenesis of endometrial carcinoma in human subjects.

Adenocarcinoma↗
PubMedDate not supplied

A rapid immunogold-silver staining for detection of bromodeoxyuridine in large numbers of plastic sections, using microwave irradiation.

A rapid and convenient method for the large scale, immunogold-silver staining (IGSS) of bromodeoxyuridine (BrdU) incorporated by S phase cells, by means of a monoclonal antibody (anti-BrdU) is described. Nineteen slides at a time can be incubated with the antibodies and the protein A-gold (PAG) in staining jars. The antibody and protein A-gold solutions could be used at least five times to incubate new batches of slides. The incubation times with these solutions were shortened by means of microwave irradiation. In this way 200 slides carrying at least 800 sections could be easily processed under the same conditions in one day, using 1.25 ml neat antibody solutions of anti-BrdU and rabbit anti-mouse. For light microscopy both plastic embedding systems: methylmethacrylate (MMA) and glycolmethacrylate (GMA) can be stained with this technique. The MMA sections, of which the plastic has to be removed before the IGSS, has the advantage of a stronger labelling intensity. The GMA plastic, which contains a cross-linking agent cannot be removed and consequently for GMA sections it is necessary to incubate the sections with a proteolytic enzyme (trypsin) before the IGSS, to reexpose the antigenic binding sides. However, the GMA sections can be allowed to air dry during the IGSS without negative effects on the morphology. This makes it possible to perform the antibody and the PAG-incubating steps on one day and to finish the IGSS the next day. In this way twice as many GMA slides can be incubated with the same antibody and PAG solutions than with MMA slides.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

The application of immunogold silver staining (IGSS) for the detection of transmissible gastroenteritis virus in fixed tissues.

Protein A-gold (PAG) and a primary porcine antiserum were used in immunogold silver staining (IGSS) for the detection of transmissible gastroenteritis virus (TGEV) in formalin-fixed paraffin-embedded tissue sections of small intestine originating from infected pigs. Immunogold electron microscopy was used to evaluate the reactivity of the prepared PAG marker with the specific porcine TGEV antiserum. Gold particles were closely associated with single virions and immune aggregates of TGEV. When IGSS, using PAG as the marker, was applied to tissue sections, dark staining of TGEV-infected villous enterocytes was observed. Background was low, allowing good visualization by light microscopy of the distribution of viral antigen. Two other gold conjugates, protein A/G-gold (PA/GG) and protein G-gold (PGG), were tested in IGSS. The labeling with PA/GG was comparable to that obtained with PAG. However, no staining was observed when PGG was used. The use of IGSS and PAG offers advantages and may represent a useful technique for the detection of other viral pathogens.

Animals↗
PubMed1990

[Application of silver acetate autometallography in histopathology: a new detection method for use in immunogold silver staining, lectin histochemistry and in situ hybridization].

Modern histochemical techniques allow the specific detection of tissue constituents in situ. Routinely formalin fixed, paraffin embedded tissues may present problems to the pathologist since destruction of substances can lead to false negative results. Immunogold-silver staining (IGSS) can be a way to overcome some of these problems. A highly efficient method allows silver enhancement to be carried out without the necessity of a dark box (silver acetate autometallography). To test the new modification, antisera to a variety of antigens were used on paraffin and semithin resin sections. The system was also tested for its applicability in multiple immunostaining techniques and for the in situ hybridisation of viral DNA and for demonstration of carbohydrates by lectin histochemistry. It showed a very high detection efficiency with low background staining.

Acetates↗
PubMedDate not supplied

Spermatocyte chromosome analysis of Helicella virgata (Pulmonata: Helicidae): silver-stained and C-banded chromosomes.

Chromosome numbers of the snail Helicella virgata from the fields of Castellammare del Golfo (Sicily) are n = 26 and 2n = 52. Silver-staining analyses of testicular cells suggest that both mitotic and meiotic chromosomes are involved in nucleolus organization. A within-individual variability in NOR-banding pattern is present in each of the 20 specimens analyzed.

Animals↗
PubMed1993

The use of dot-immunogold-silver staining (dot-IGSS), dot-ELISA and dot-IGSS to detect serum antibodies from clonorchiasis patients: a comparative study.

We report the use of immunogold-silver staining (IGSS), dot-ELISA and dot-IGSS methods in the study of clonorchiasis in China. These methods were employed to detect the antibody in sera from 40 clonorchiasis patients. The positive rates were 100%, 90.0% and 95.0%, respectively. When the three methods were used to examine 40 normal sera, the negative rates were 100%, 97.5% and 97.5%, respectively. These results suggest that IGSS, dot-ELISA and dot-IGSS are highly specific and sensitive in detecting anti-Clonorchis antibody in patients.

Antibodies, Helminth↗
PubMed1989

Morphological and functional techniques for assessing endothelial integrity: the use of Evans blue dye, silver stains, and endothelial derived relaxing factor.

The endothelium is an important aspect of vascular function and pathology. Simple and reliable methods for assessing the presence or absence of endothelium in vascular specimens are presented. Intravenous Evans blue dye that stains endothelial denuded vessel walls in recommended for the macroscopic visualization of endothelial injury in gross intact arterial specimens. Scanning electron microscopy (SEM) is often used for investigating microscopic endothelial injury, but the differentiation of endothelial cells from underlying vessel wall structures can be difficult. By demarcating endothelial cell borders, silver stains allow for better endothelial identification by SEM. A third technique describes assessing endothelial integrity by how well it functions and involves the selective production of endothelium derived relaxing factor. As cardiovascular surgeons become more involved in basic vascular research, a review of these techniques should be appropriate and helpful.

Animals↗