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PubMed1989

Application of immunogold-silver staining and immunoenzymatic methods in multiple labelling of human pancreatic Langerhans islet cells.

The use of immunogold-silver staining (IGSS) combined with immunoperoxidase and/or immunoalkaline phosphatase methods for the simultaneous demonstration of pancreatic islet cell hormones on routinely fixed paraffin-embedded human tissue sections was examined. If IGSS was applied first, the black colour of silver-enhanced colloidal gold on doubly immunostained sections contrasted with the colours of most of the chromogens used generally in the 2 immunoenzymatic methods. If IGSS was followed by immunoalkaline phosphatase and immunoperoxidase techniques in optional sequence, 3 different hormone-containing cell types could be stained simultaneously without non-specific cross-reactions. IGSS and immunoalkaline phosphatase methods, together with 2 kinds of non-cross-reacting immunoperoxidase systems, permitted the detection of 4 distinct antigens on the same tissue section. Multiple immunohistochemical labelling of the endocrine pancreas provides an opportunity for the correct and rapid analysis of the topographic and morphometric relationships between different hormone-producing cell populations under both normal and pathological conditions. IGSS is of great potential for the simultaneous immunolabelling of antigens situated within separate cells.

Chronic Disease↗

PubMed2005

Silver stainings distinguish Lewy bodies and glial cytoplasmic inclusions: comparison between Gallyas-Braak and Campbell-Switzer methods.

Lewy bodies (LBs) of idiopathic Parkinson's disease and glial cytoplasmic inclusions (GCIs) of multiple system atrophy are pathological deposits both composed of phosphorylated alpha-synuclein woven into different filaments. Although both LBs and GCIs are considered to be hallmarks for each independent synucleinopathy, until now they could not be clearly distinguished on the basis of their biochemical or immunohistochemical features. We have examined possible differences in their argyrophilic features and their relation to synuclein-like or ubiquitin-like immunoreactivity (IR). Pairs of mirror sections from different brain areas were triple-fluorolabeled with an anti-alpha-synuclein antibody, an anti-ubiquitin antibody and thiazin red (TR), a fluorochrome that labels fibrillary structures such as Lewy bodies or neurofibrillary tangles. One of the paired sections was subsequently stained using the Campbell-Switzer method (CS), and the other by the Gallyas-Braak method (GB). By comparing of the same microscopic field on the paired fluorolabeled sections, subsequently silver-stained with either CS or GB, five different profiles of each structure could be determined: alpha-synuclein-like IR, ubiquitin-like IR, affinity to TR, argyrophilia with CS or GB. GCIs exhibited argyrophilia with both CS and GB but lacked affinity to TR. In contrast, LBs exhibited argyrophilia with CS but not with GB and some affinity to TR. These disease-specific profiles of argyrophilia were consistent, and were not influenced by areas or cases examined. Although immunohistochemical features of LBs and GCIs were similar in exhibiting IR for alpha-synuclein and ubiquitin, the contrast in their argyrophilic profiles may indicate possible differences in the molecular composition or conformation of alpha-synuclein. Even though these empirical differences still remain to be explained, awareness of this clear distinction is potentially of diagnostic and pathological relevance.

Aged↗

PubMed1991

Silver staining of senile plaques and neurofibrillary tangles in paraffin sections. A simple and effective method.

A one-step silver staining method using a protective colloidal developer of gelatin and formic acid was originally established for demonstration of argyrophilic nucleolus organizer regions by controlled reduction of silver. We describe here a new application of this silver technique that can easily be performed to demonstrate senile plaques (SP) and neurofibrillary tangles (NFT) on paraffin sections. Preliminary results in ten cases of senile dementia of Alzheimer type and five cases of amyloid congophilic angiopathy showed a reliable demonstration of amyloid and neuritic type SP as well as NFT in all 15 cases. In addition, deposits of perivascular amyloid and areas of fibrillar amyloid, the diffuse type of senile plaques, were revealed. The success seems to depend particularly on the low concentration of formic acid in the developer, which might be the responsible agent for the careful revealing of buried argyrophilic structures in SP and NFT. The staining features were quite similar to those with anti-beta and anti-tau immunostaining. This result suggests a high specificity of this method for extracellular and intraneuronal cerebral amyloid. The detailed staining of delicate morphological structures points to a high sensitivity for cerebral amyloids, senile plaques and neurofibrillary tangles, respectively, by this simple and inexpensive method.

Aged↗

PubMed2003

Micronuclei, nuclear lesions and interphase silver-stained nucleolar organizer regions (AgNORs) as cyto-genotoxicity indicators in Oreochromis niloticus exposed to textile mill effluent.

In this study, cyto-genotoxic effects of a textile mill effluent on fish Oreochromis niloticus were investigated using the micronucleus (MN) test and methods to analyze interphase silver-stained nucleolar organizer regions (AgNORs). Fishes were exposed to three different concentrations of textile mill effluent (5, 10 and 20%(v/v)) for 3, 6 and 9 days. Cyclophosphamide (2mg/l) was used as a positive control. Micronucleus frequencies were examined in peripheral blood erythrocytes and gill cells. Nuclear abnormalities (NA) other than micronuclei such as binuclei, lobed nuclei, blebbed nuclei and notched nuclei were also evaluated in peripheral erythrocytes. Interphase AgNOR parameters were examined in epithelial cells obtained from the edge of caudal fins after 90 and 180min of exposure. As a result, dose-dependent increases in the frequencies of micronuclei and other NA in erythrocytes were observed. MN frequencies in gill cells also significantly increased, while the interphase AgNOR parameters in fin cells decreased, as a result of textile effluent and cylophosphamide treatments.

Animals↗

PubMedDate not supplied

Quantification of silver-staining nucleolar organizer region in epithelial cells of tongue of mice after exposure to, or intake of, alcohol.

The purpose of the current study was to evaluate the effect of alcohol on the proliferative activity of epithelial cells in the lingual mucosa of mice by means of silver-staining nucleolar organizer region (AgNOR) count and area measurements. Forty-eight CF1 mice were separated into three groups. The test groups were submitted to topical exposure to, or intake of, 40% (volume/volume) ethyl alcohol. Biopsy specimens were collected from the middle third of the dorsal tongue at 0, 6, and 12 months, and samples were stained according to the AgNOR technique. Mean number and mean area of AgNOR per nucleus were calculated for 50 basal layer cells and 50 intermediate layer cells. Increases in mean number and mean area of AgNOR per nucleus in intermediate cells were observed at 12 months in the alcohol intake group (P < .05). Results showed that intake of 40% alcohol increased epithelial cell proliferation in the dorsal surface of lingual mucosa.

Animals↗

PubMed1990

Discrimination of fetal bovine serum from other sera by silver staining or lectin blotting of SDS/PAGE-separated serum proteins.

In order to develop a methodology to discriminate fetal bovine serum from other sera with which it might be contaminated, proteins from fetal bovine, newborn calf, colostrum-free newborn, and calf sera were separated by SDS/PAGE and analyzed by silver staining of the gels or lectin blotting, using concanavalin A (Con A), following transfer of the proteins to nitrocellulose. A high molecular weight Con A-reactive glycoprotein of an apparent molecular mass greater than 200,000 daltons was present in newborn, colostrum-free, and calf sera, but absent or at a significantly lower concentration in fetal serum. These two methods accurately and reliably identified contamination of fetal serum with other sera in a series of blind studies on unknown, coded samples. As little as 1% colostrum-free serum in a batch of fetal serum can be detected by either procedure.

Animals↗

PubMed1986

Nucleolar activity after 3-methylcholanthrene treatment of rat liver cells studied by silver staining procedure.

The influence of a single dose of 3-methylcholanthrene (3-MC) was studied in nucleoli of young rat liver cells by means of conventional and ultracytochemical methods. The nucleolar activity was stimulated in our experimental conditions: the appearance of the fibrillar centres in the liver cell nucleoli as well as the silver staining protein content of the fibrillar centres and the dense fibrillar component were increased by 3-MC. The results suggest that the activity of ribosomal genes was increased following 3-MC treatment.

Animals↗

PubMed1997

Villous cytotrophoblast proliferating potential in complete and partial hydatidiform mole: diagnostic value of silver-stained nucleolar organizer region (AgNOR)-associated proteins.

Aside from their typical morphologic features, complete (CHM) and partial hydatidiform mole (PHM) are characterized by variable trophoblastic proliferation and/or atypia. CHM and PHM usually present little diagnostic difficulty. However, some may be extremely difficult to distinguish by morphologic features alone. Therefore, we investigated the diagnostic value of silver-stained nucleolar organizer region (AgNOR)-associated proteins in cytotrophblasts as compared to cytogenetic features of nine CHM, nine PHM and six non-molar spontaneous embryonic abortions (controls), as well as of two suspected CHM and two histologically suspected PHM. Tissue sections were submitted to autoclave pretreatment and to silver colloid solution. The proliferating potential of cytotrophoblasts was determined by the analysis of mean number and mean area of AgNORs per nucleus using a PC-based image analysis system. Mean values of AgNOR parameters were significantly different from each other (p < 0.001). Each of the four cases of tentative diagnosis could be assigned to the corresponding group of examined trophoblastic lesions. The evaluation of AgNORs in cytotrophoblasts contributes to a reliable discrimination of CHM and PHM; this fairly simple and economical method could serve as an useful addition to conventional methods of diagnosis in gestational trophoblastic disease.

Cell Division↗

PubMed2006

Improved mass spectrometry compatibility is afforded by ammoniacal silver staining.

Sequence coverage in MS analysis of protein digestion-derived peptides is a key issue for detailed characterization of proteins or identification at low quantities. In gel-based proteomics studies, the sequence coverage greatly depends on the protein detection method. It is shown here that ammoniacal silver detection methods offer improved sequence coverage over standard silver nitrate methods, while keeping the high sensitivity of silver staining. With the development of 2D-PAGE-based proteomics, another burden is placed on the detection methods used for protein detection on 2-D-gels. Besides the classical requirements of linearity, sensitivity, and homogeneity from one protein to another, detection methods must now take into account another aspect, namely their compatibility with MS. This compatibility is evidenced by two different and complementary aspects, which are (i) the absence of adducts and artefactual modifications on the peptides obtained after protease digestion of a protein detected and digested in - gel, and (ii) the quantitative yield of peptides recovered after digestion and analyzed by the mass spectrometer. While this quantitative yield is not very important per se, it is however a crucial parameter as it strongly influences the S/N of the mass spectrum and thus the number of peptides that can be detected from a given protein input, especially at low protein amounts. This influences in turn the sequence coverage and thus the detail of the analysis provided by the mass spectrometer.

Ammonia↗

PubMedDate not supplied

[The use of a silver staining method in the diagnosis of the late skin lesions in ixodid tick-borne borreliosis in man].

The borreliosis etiology was verified in 60% cases of the morphea, 100% cases of the atrophoderma of Pasini-Pierini and 100% cases of anetoderma by means of IFA with Ip-21 antigen (B. afzelii) and silver staining after Levadity. Borreliae were located near the capillaries, muscular and areolar tissue fibers of the derm and near the granulated cells of epidermis.

Animals↗

PubMed1983

Immunogold-silver staining: new method of immunostaining with enhanced sensitivity.

A new method for demonstrating antigens in paraffin sections of formol sublimate-fixed tissue is described that utilizes an "indirect" immunohistological technique employing immunoglobulin adsorbed to colloidal gold as the secondary antiserum. The gold particles introduced to antigenic sites are revealed by a silver precipitation reaction. This technique, the immunogold-silver staining method, is of much enhanced sensitivity (up to 200-fold) as compared with standard immunoperoxidase and immunogold staining methods. The results have been confirmed in a study of immunoglobulins in reactive human tonsil. The use of this new method for double immunolabeling is also described.

Animals↗

PubMed1991

Technical considerations in evaluating the endothelial integrity of rat aortic preparations with silver staining.

Assessment of endothelial integrity is an obligatory step in many pharmacological studies. Integrity of endothelium is affected by manipulations performed during the removal and cleaning of the vessel and by some of the silver-staining techniques utilized for demonstrating interendothelial junctions. When aortas were cleaned of periadventitial tissue in cold Tris-saline (once separated from the animal) by untrained personnel, only 45% of the endothelium was preserved. When cleaning was performed in situ by trained personnel while flushing with cold Krebs-Ringer-6% albumin, over 95% was left intact. AgNO3-staining performed before fixation produced a 50% loss of endothelium when using NH4Br and (NH4)2S as developers. AgNO3-staining performed after fixation produced over 95% recuperation of endothelium when 2% glutaraldehyde, 150 mM NaCl, 40 mM phosphate buffer, pH 7.4, were utilized as initial fixative, NH4Br and (NH4)2S being equally effective as developers. Chloride ions were necessary to intensify silver lines. Several patterns of deendothelization were produced by mechanical and chemical injury with saponin, NH4Br and (NH4)2S. In all cases, hematoxylin staining was employed as an auxiliary technique to interpret images of injured endothelium. Presence of albumin protected the endothelium from mechanical damage.

Animals↗

PubMed1995

Characterization of Dasypyrum villosum (L.) candargy chromosomal chromatin by means of in situ restriction endonucleases, fluorochromes, silver staining and C-banding.

The distribution of C-banded heterochromatin was determined in an inbred line of Dasypyrum villosum. Practically no difference in chromosome morphology or band distribution could be observed within the chromosomes of the same pair. Heterochromatin bands, revealed by Giemsa banding, were characterized by means of their differential reaction to fluorochromes, silver staining and in situ digestion with different restriction endonucleases. The results clearly indicate that in D. villosum two different classes of heterochromatin with different chromosomal local-ization exist: one is evidenced by both C-banding and DAPI staining and has mainly telomeric distribution, the other is evidenced only by C-banding and has mainly centromeric distribution.

Chromosome Banding↗

PubMed1987

[Detection of beta 2-transferrin with agarose gel electrophoresis, immunofixation and silver staining in cerebrospinal fluid, secretions and other body fluids].

A method is described for the detection of beta 2-transferrin (asialotransferrin) in 1 microliter cerebrospinal fluid. Owing to its low content of sialic acid, beta 2-transferrin migrates more slowly than the main transferrin component (beta 1-transferrin) in electrophoresis. The high sensitivity and specificity of the test depend on immunofixation of the electrophoretically separated transferrin in the agarose gel, and on the visualization of the immune complex by staining with alkaline silver nitrate solution. Since beta 2-transferrin occurs practically only in cerebrospinal fluid and not in other body fluids, its detection in secretions from the nose or ear can be used for the diagnosis of rhinoor otoliquorrhoea. Even under unfavourable conditions, 10% cerebrospinal fluid can be detected in a secretion. Operator time for investigation of a secretion sample is 110 minutes and the analysis time is 5 hours. The direct costs are 95 German marks. Forty two secretions have been investigated so far. Eighteen were shown to contain cerebrospinal fluid, and this was confirmed by subsequent operative findings on the patients.

Asialoglycoproteins↗

PubMedDate not supplied

Two-dimensional electrophoresis and densitometric analysis of solubilized bovine sperm plasma membrane proteins detected by silver staining and radioiodination.

Bovine sperm plasma membranes were extracted with deoxycholate and subjected to two-dimensional gel electrophoresis. Proteins were visualized either by silver staining or autoradiography using 125I. When 1.5-2.0 micrograms of protein extract was applied to the first-dimension get, 250 spots could be detected by autoradiography. Thirty micrograms of protein was required to obtain spot visualization using silver strain, revealing more than 500 spots on the gel. These data establish the limit under which two-dimensional electrophoresis can be used in conjunction with flow cytometric sorting of sperm for the analysis of possible sex-specific membrane differences. The necessity of orienting sperm as they past the laser in the cell sorting system reduces throughput; thus, the number of sperm sorted within a given time is limited. It is suggested that autoradiography will allow the flow cytometer to be used in a time-efficient manner. Longer sorting times would be required to obtain sufficient sample to analyze total protein composition.

Animals↗

PubMed1988

Differential ribosomal gene responsiveness to human growth hormone is visualized by selective silver staining.

Differential activity of rRNA gene clusters following growth-hormone administration has been demonstrated in cultured lymphocytes from subjects with different genetic backgrounds, i.e., with or without in vivo peripheral responsiveness to the hormone. The influence of different culture conditions on ribosomal gene responsiveness was also tested. Ribosomal gene activity was evaluated by selective silver staining of nucleolus organizing regions. The results show that hormone-induced enhancement of transcriptional activity requires both genetically determined cell responsiveness and environmentally determined permissive factors.

DNA, Ribosomal↗

PubMed1994

Comparative study of Dot-immunogold silver staining and Dot-ELISA for the detection of serum antibodies against Wuchereria bancrofti.

Dot-immunogold silver staining (Dot-IGSS) and Dot-ELISA, using the soluble antigen of Brugia malayi, were employed to detect anti-Wuchereria bancrofti antibodies in 50 cases of Wuchereria bancrofti microfilaremia. The positive rates were 100% and 90% in Dot-IGSS and Dot-ELISA respectively. The average titer in the 45 positive cases was 1:184 (1:10-1:2560) for Dot-IGSS and 1:150 (1:10-1:2560) for Dot-ELISA, with 30 cases showing the same titer in both tests, 13 cases showing higher titer in Dot-IGSS than in Dot-ELISA and 2 cases in the former showing lower titers than in the latter. There was a linear relationship between the titers of antibodies detected by Dot-IGSS and by Dot-ELISA (r = 0.8443). Dot-IGSS, similar to Dot-ELISA, is easy to carry out and the result is easy to read. It is seen that Dot-IGSS is highly sensitive and specific and is practicable for immunodiagnosis and surveillance of filariasis.

Animals↗

PubMed1987

Human ribosomal DNA fragments amplified in hamster cells are transcribed only by RNA polymerase II and are not silver stained.

Cloned human rRNA gene fragments that included the promoter region were introduced into Chinese hamster dihydrofolate reductase-deficient (dhfr-) cells by cotransformation with a dhfr minigene and amplified by selection for methotrexate resistance. The human ribosomal DNA was transcribed by RNA polymerase II, not RNA polymerase I or III. The metaphase chromosome regions containing the transcriptionally active human ribosomal DNA failed to show silver staining.

Animals↗