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PubMed1996

Dot-immunogold-silver staining in the diagnosis of cysticercosis.

Dot-immunogold-silver staining (Dot-IGSS) and Dot-ELISA were used, with Taenia solium cyst fluid antigen, to detect specific antibodies in the sera of patients with cysticercosis. All 40 confirmed cases of cysticercosis were found to be positive when tested by 2 methods. The average titer of the sera detected by Dot-IGSS was 1:27,470, which was significantly higher than that detected by Dot-ELISA. The false positive rates were 2% and 4% in 50 healthy controls for Dot-IGSS and Dot-ELISA, respectively, with a low titer of 1:20.

Antibodies, Helminth↗
PubMed1980

Alcoholic Bouin fixation of insect nervous systems for bodian silver staining. III. A shortened, single impregnation method.

Satisfactory Bodian silver staining of paraffin wax sections of both locust (Schistocerca gregaria) and cockroach (Periplaneta americana) central nerve tissue can be obtained with only one impregnation, instead of the usual two, by the following modified procedure. Freshly dissected ganglia are fixed in an improved synthetic alcoholic Bouin (40% formaldehyde 0-15: ethanol 25: acetic acid 5: picric acid 0.5: either ethyl acetate 5 and diethoxymethane 15, or ethyl acetate 25: distilled water to 100). Formaldehyde content governs intensity of glial staining (little or none without formaldehyde) and the mixture with more ethyl acetate substituted for diethoxymethane gives more intense staining overall. Sections are impregnated once only, overnight, in 2% Protargol solution brought to about pH 8.4 with ammonium hydroxide and containing 1.3 g of copper per 65 ml. Depending on fixative composition, species, section thickness and contrast desired between nerve fibers and background, the subsequent distilled water rinse is shortened or omitted and sections are developed in 1% hydroquinone with sodium sulfite content reduced (to 2.5-4% Na2SO3.7H2O) for thinner (10 micrometer) sections but normal (10%) for thicker (20 micrometer) ones. Sections are finally washed, gold intensified, treated with sodium thiosulfate and dehydrated, cleared and mounted as usual. Results are slightly lighter than with normal double impregnation but entirely suitable for studies of neuroanatomy.

Animals↗
PubMed1995

Diagnostic relevance of silver stained nucleolar organizer region (AgNORs) in benign and malignant breast lesions.

The present study is planned to assess the diagnostic relevance of silver stained nucleolar organizer regions (NORs) in discriminating benign from malignant lesions of the breast and to ascertain the interobserver variability in the enumeration of AgNOR counts. Forty four specimens of benign and malignant breast tissues were studied and mean number of silver stained nucleolar organizer regions (MNORA) have been counted in paraffin sections. Malignant lesions have a higher number of MNORA than benign lesions. These results also showed correlation between the AgNOR counts and size of the tumor, axillary lymph node status and age of the patient. Our results also indicate a close agreement between the two observers with limits of agreement. However, AgNOR counts are of little diagnostic importance since a broad zone of overlap exists between benign and malignant breast diseases despite significant higher mean counts in malignant lesions. AgNOR sequence could perhaps be of use to differentiate those two histological indistinguishable lesions. The tumor size and axillary lymph node involvement suggested to its prognostic significance also. Interobserver agreement further suggests the utility of AgNOR counts as diagnostic or prognostic discriminants in breast malignancies.

Breast Diseases↗
PubMed1992

Apolipoprotein E phenotyping by isoelectric focusing in immobilized pH gradients and silver staining.

A simple and high resolution procedure of apoprotein E (apo E) phenotyping by isoelectric focusing with immobilized pH gradients and silver staining is described. This method needs delipidated very low density lipoproteins (isolated from 1 mL of serum) but obviates immunoblotting as well as neuraminidase treatment in routine applications because the sialylated forms are clearly separated. Immunoblotting (with polyclonal and monoclonal anti-apo E antiserum), cysteamine and neuraminidase treatment, and pI markers allowed the localization of three main alleles, xi 2, xi 3, xi 4 and the detection of variants or rare alleles (6/450 determinations). The serum amyloid A (SAA) apolipoproteins (SAA1,SAA2) could be characterized unequivocally (especially with E3 and E4). Silver staining proved more sensitive than Coomassie Brilliant Blue and needs only 5 micrograms of protein in the sample. The results of 403 normo-or hyperlipidemic patients are shown. In the group of 191 normolipidemic patients (cholesterol less than 6.40 mmol/L triglycerides less than 2 mmol/L), the relative frequency of the xi 3 allele (0.83) is higher than in other reports on Caucasians (about 0.77) whereas the xi 4 allele is lower. As previously described, we find a high frequency of the 4/3 phenotype in hypercholesterolemia and 3/2 in hypertriglyceridemia. The high frequency of the E2/E2 phenotype, usually associated with hyperlipidemia, and variants in complex hypertriglyceridemia makes the apo E phenotyping necessary in many cases of dyslipidemias.

Acrylamides↗
PubMedDate not supplied

Application of silver stains to cytologic specimens of neuroendocrine tumors metastatic to the liver.

Cytologic specimens of neuroendocrine tumors metastatic to the liver were examined with regard to their silver staining properties after the application of argentaffin and argyrophil staining techniques (Masson, Grimelius and Sevier-Munger). In tumors with a content of serotonin (small intestine carcinoids), the presence of this substance was demonstrated cytologically as an argentaffin reaction in individual tumor cells; however, formalin fixation was a prerequisite for positive staining. Melanin in malignant melanoma cells displayed a positive argentaffin reaction, irrespective of the fixation used (air drying, formalin, Bouin's fluid or acetone-alcohol). Thus, serotonin and melanin can be distinguished in cytologic samples of neuroendocrine tumors by the use of the Masson argentaffin reaction with different fixatives. The nonargentaffin-positive neuroendocrine tumor cells were weakly stained or unreactive with the Grimelius argyrophil technique. The Sevier-Munger argyrophil technique was negative or gave a disturbing nonspecific background staining reaction that was difficult to interpret in the cytologic samples. Thus, the Grimelius method appears to be the most useful silver stain for identifying neuroendocrine tumor cells in cytologic material, irrespective of their hormone content, since both argentaffin-positive and argentaffin-negative cell samples were stained at least to some degree.

Adenocarcinoma↗
PubMed1988

Light microscopic localization of alkaline phosphatase in fetal bovine bone using immunoperoxidase and immunogold-silver staining procedures.

We localized alkaline phosphatase in the metaphyses of fetal bovine tibial bone by use of avidin-biotin-immunoperoxidase and immunogold-silver staining procedures. Low melting-point, paraffin-embedded sections of periodate lysine-paraformaldehyde-fixed undecalcified bone were used for immunostaining. We suggest that the combination of intact embryonic bone with this fixative and the immunohistochemical procedures used in this study may have helped to preserve antigenicity and thus to improve the efficiency of immunolabeling. Similar patterns of alkaline phosphatase localization were produced by the immunoperoxidase and immunogold-silver staining methods. The latter, although free of immunoreagents such as diaminobenzidine, must be monitored closely to avoid nonspecific staining during the silver enhancement procedure. Both methods revealed a concentration of the enzyme in osteoblasts and in areas of osteoid that lined the bone trabeculae. The results support the findings of earlier enzyme cytochemical studies in which osteoblasts were shown to have significant alkaline phosphatase activity.

Alkaline Phosphatase↗
PubMed1986

An immunogold-silver staining method for detection of cell-surface antigens in light microscopy.

An immunogold-silver staining technique for detection of cell-surface antigens in cell suspensions was developed. Leukocyte cell suspensions were first incubated with monoclonal antibodies directed against cell-surface antigens and then with colloidal gold-labeled goat anti-mouse antibodies. Cytocentrifuge preparations of the cell suspensions were immersed in a physical developer containing silver lactate and hydroquinone as reducing substance. The preparations were then counterstained and mounted. In light microscopy, cells reacting with the monoclonal antibodies showed dark granules on their surface membrane. An optimal morphology, as revealed by a May-Grünwald-Giemsa counterstain, permitted accurate cell identification. The labeling was influenced by the gold particle diameter and the concentration of the gold reagents, by the duration of incubation in the physical developer, and by the composition and temperature of this medium. The T-cell subsets enumerated with this method in the peripheral blood of normal adults were identical to those found with other methods. The sensitivity of the technique was comparable with that of immunofluorescence microscopy. This immunogold-silver staining procedure proved to be a reliable tool for detection of cell-surface antigens in light microscopy.

Antibodies, Monoclonal↗
PubMed1993

Expression of silver-stained nucleolar organizer regions is coupled to cell cycle in rat thymic cells.

To directly analyze the relationship between the expression of silver-stained nucleolar organizer regions (AgNORs) and cell proliferation, thymic cells from newborn rats were separately sorted at the G0-G1-phase and early-mid and late-mid S-phase of the cell cycle according to their DNA content. Different AgNOR-derived parameters (mean area and numbers of AgNORs per cell and mean AgNOR-particle area) were evaluated after silver staining of cytospins. A linear correlation was observed between the mean area and numbers of AgNORs per cell, both parameters increasing progressively from G0-G1-phase to early-mid and late-mid S-phase. An increase of the mean AgNOR-particle area was also seen between G0-G1 and S-phase but this was not significant along the S-phase. A bias on the selection of S-phase cells linked to intrathymic maturation can be ruled out as S-phase cells labeled with bromodeoxyuridine were found throughout the thymus, and, moreover, the analysis of the frequency distribution of nuclear area did not show a bimodal pattern. It is concluded that the expression of AgNORs--evaluated as AgNOR area/cell or AgNOR numbers/cell--is causally or indirectly coupled to DNA synthesis and, thus, AgNORs can be considered as a cell proliferation marker.

Animals↗
PubMed1995

[The squash technique for silver staining of argyrophilic nucleolar organizer regions on bladder tumors: comparison with paraffin embedded sections].

We examined nucleolar organizer region (NOR) indices on 13 bladder tumors on both squash-prepared specimens and paraffin embedded specimens stained by silver-colloid method. It was noted that; 1. The nuclear size was enlarged by squashing, 2. Aggregated NOR dots were dispersed, 3. Uniformity of microscopic phase of the dots was obtained, 4. Squash specimens had more precise countabilities than the paraffin embedded sections for avoidance of nuclear resection. NOR indices (mean +/- S.D.) of those bladder tumors on squash-preparation and paraffin section were 8.64 +/- 4.32 and 6.13 +/- 1.58, respectively, and the former showed good correlation with the tumor stage. When we prepare the squashed specimen from the surgically resected bladder tumor tissue, embedding and sectioning processes which are inevitable for preparation of paraffin embedded section, could be eliminated. Thus, squash-smear technique was considered to be better than the method using paraffin embedded section on the point of shorter duration of examination and accurate countability of the NOR dots.

Aged↗
PubMed2003

Evaluation of individual protein errors in silver-stained two-dimensional gels.

The relationship between spot volume and variation for all protein spots observed on large format 2D gels when utilising silver stain technology and a model system based on mammalian NSO cell extracts is reported. By running multiple gels we have shown that the reproducibility of data generated in this way is dependent on individual protein spot volumes, which in turn are directly correlated with the coefficient of variation. The coefficients of variation across all observed protein spots were highest for low abundant proteins which are the primary contributors to process error, and lowest for more abundant proteins. Using the relationship between spot volume and coefficient of variation we show it is necessary to calculate variation for individual protein spot volumes. The inherent limitations of silver staining therefore mean that errors in individual protein spot volumes must be considered when assessing significant changes in protein spot volume and not global error.

Animals↗
PubMed1979

Chromosome core structure revealed by silver staining.

Chromosomes were subjected to either prolonged hypotonic solution pretreatment or aging. Both conditions greatly loosened and dispersed the overlying epichromatin from the central chromosome core structure. This was followed by silver staining and examination with bright-field microscopy. The chromosome core selectively reduced the silver and stained black while the surrounding epichromatin stained yellow. A single core was seen extending the length of each chromatid. Nucleolus organizer regions appeared to be attached to the core, while kinetochores seemed to be specialized regions of the core itself. Cytochemical tests indicated that the core component(s) responsible for silver staining was non-histone protein(s).

Adenocarcinoma↗
PubMed1988

Selective silver-staining methods for RNA and proteins in the same polyacrylamide gels.

Two silver-staining methods for selective and ultrasensitive detection of RNAs and proteins in the same polyacrylamide gels were developed, both derived from procedures recommended for protein staining. The first, a double-staining technic with Coomassie brilliant blue and ammoniacal silver, allows visualization of RNAs as negative bands and proteins as dark brown bands. The second is also a double-staining technique, but uses silver in both steps. This second method develops the RNA bands first and then the protein bands. These techniques, especially the second, permit characterization of the different components of ribonucleoproteic complexes in the same electrophoresis gels.

Electrophoresis, Polyacrylamide Gel↗
PubMed1976

An improved technique for selective silver staining of nucleolar organizer regions in human chromosomes.

A reliable technique for staining human chromosomal nucleolar organizers (NOR's) with silver solutions is described. The NOR's can be selectively stained dark brown by silver solutions leaving the chromosome arms unstained and available for counterstaining with orcein or Giemsa dyes. Unequivocal identification of chromosome pairs bearing NOR's can be achieved using fluorescent banding techniques followed by silver staining. The silver staining procedure for NOR's was simplified and standardized through control of the chemical and physical conditions during silver impregnation and developing.

Chromosomes↗
PubMed1990

Silver staining of nucleolar organizer region associated proteins using polyethylene glycol as the protective colloidal developer.

A simple modification to the silver staining technique for the demonstration of nucleolar organizer region associated proteins is described. Polyethylene glycol 20,000 is used instead of gelatin as the colloidal developer. This modified technique remains a one stage procedure that is quick and easy to perform. It results in reduced precipitate and less non-specific staining with specimens in which it had been previously difficult to demonstrate these intranuclear silver staining structures.

Animals↗
PubMed1997

Heparins, low-molecular-weight heparins, and other glycosaminoglycans analyzed by agarose gel electrophoresis and azure A-silver staining.

A sensitive, nonradioactive azure A-silver staining method combining agarose gel electrophoresis was established and evaluated. Unfractionated heparins (UFHs), low-molecular-weight heparins (LMWHs), heparan sulfate (HS), chondroitin sulfate A (CSA), dermatan sulfate (DS), keratan sulfate (KS), and hyaluronic acid (HA) were analyzed. The detection limit of the method was 0.5 ng for heparin, LMWH, HA, CSA, and DS, 2 ng for KS, and 6 ng for HA in the 2-microliter sample volume. Dilution curves demonstrated linear correlation between the logarithm of the concentration of glycosaminoglycans (GAGs) and their optical absorbance at 548 nm. The linear ranges were 1 to 500 ng/microliter for heparins, LMWHs, HS, DS, and CSA, 3 to 500 ng/microliter for KS, and 8 to 500 ng/microliter for HA. GAGs have their characteristic migration patterns and their Rf value decreased from CSA to KS, DS, HS, heparin, and HA. The differences were described for heparins and LMWHs. LMWHs migrated faster and displayed broader bands than unfractionated heparins. It was also observed that some unfractionated heparins contained low sulfated GAGs as contamination, which seemed to be DS as judged by their migration patterns.

Azure Stains↗
PubMed1991

Leukemia and lymphoma immunophenotyping in cell smears with immunogold-silver staining.

The potential of the immunogold-silver staining (IGSS) technique for immunophenotyping leukemia and lymphoma cells in cell smears was examined. Peripheral blood, bone marrow aspirates, lymph node biopsy specimens, fine-needle aspirates, and biologic fluids of 83 patients with acute or chronic leukemias, non-Hodgkin's lymphomas, or Hodgkin's disease were labeled. Cell smears, cytocentrifuge preparations, or imprints were fixed, incubated with the reagents, and counterstained with May-Grünwald-Giemsa. Stable immunostaining and good morphologic characteristics allowed accurate cell identification and rapid enumeration of the positive cells. The immunophenotypes obtained with the use of 35 monoclonal antibodies with different specificities were similar to those determined by flow cytometry or immunohistochemical studies on the same samples. This IGSS method was especially useful for the examination of poor samples or complex cell suspensions with rare malignant cells. It could be an alternative to the immunoenzyme methods that generally are used for this purpose.

Antibodies, Monoclonal↗