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Staining of polyacrylamide gels with methylene blue prior to silver staining increases band resolution and sensitivity. This method permits resolution of multiple bands less than 1 mm apart, and is able to detect bands containing only 100 pg of RNA.
Silver staining of nucleolar organizer regions (NORs) and their subsequent quantification by image analysis are used increasingly in human pathological specimens and experimental models. Because certain conditions determined by the type of tissue and/or its fixation render AgNOR segmentation for image analysis difficult due to insufficient contrast or nonspecific silver precipitation, we propose three improvements to the original technique to overcome these difficulties. Pretreatment with 7% nitric acid produced very distinct dark brown images of AgNORs on a yellow background. The gradient of background colors allowed easy discrimination of nucleolar, nuclear and cytoplasmic structures. Seven morphometric parameters related to number, size and shape of AgNORs were evaluated quantitatively by image analysis on sections pretreated with nitric acid and on adjacent sections treated with citrate buffer in a wet autoclave according to the most widely accepted method for image analysis of AgNOR. Both methods yielded similar results. A second improvement was achieved by coating the slides with 7% celloidin solution in ethyl alcohol-ether prior to AgNOR staining and acid pretreatment. This coating prevented nonspecific silver deposition on argyrophilic bacteria and other tissue debris in human vaginal smears that could make visualizing AgNOR sites difficult. Finally, placing sections face down on the staining solution prevents the formation of nonspecific silver precipitates. These procedures can be applied together or separately according to the requirements of the material to be evaluated.
Restriction endonuclease analysis of pseudorabies virus DNA has been used to study various virus strains. To make use of a rapid technique for the identification of viral strains, studies have been undertaken to facilitate purification of the DNA from viral particles present in cytoplasmic fractions. The ultrasensitive photochemical silver staining of nucleic acid, described by Beidler et al. (Analytic Biochemistry 1982: 126) has been adapted and applied to the detection of pseudorabies virus restriction fragments. In a period of 5 h more than 1 microgram of DNA can be extracted from a 25 cm2 plastic flask containing infected cells and purified without ultra centrifugation. Low molecular weight DNA was separated from high molecular weight DNA by polyacrylamide gel electrophoresis. The restriction fragments were selectively visualized by silver staining which can detect 0.025 micrograms of total pseudorabies virus DNA. The electrophoretic DNA pattern of vaccine and wild strains has been studied using these techniques and the results agree with previously published data.
Human rotaviruses (HRV) are the most important etiologic agents of acquired diarrhea in infants and young children worldwide. Therefore, the early diagnosis is essential for effective patient management and infection control. We have developed a rapid, simple technique for the diagnosis of rotavirus based on the sensitive detection of rotavirus double-stranded RNA genome segments separated in vertical agarose gels and developed by silver staining (AGE-SS). This method also has the ability to detect differences in the electrophoretic mobility of RNA bands among group C rotaviruses, reovirus, and group A rotaviruses. The results indicate that this assay is as sensitive and specific as the polyacrylamide gel electrophoresis and silver stain method (PAGE/SS) and it could be applied on large scale for the screening of stool suspected of rotaviral diarrhea. This assay does not need sophisticated equipment and the cost per sample is minimal compared with other available assays.
B-cell chronic lymphocytic leukemia (B-CLL) is typically a low-grade neoplasm with a diploid DNA index and low proliferative activity. Interleukin-2 receptor (IL-2R/CD25) positivity often indicates increased proliferative activity and activation in both T and B lymphocytes. The argyrophilic nucleolar organizer regions (AgNORs) are loops of DNA identified by a silver staining technique and have been correlated with ploidy and proliferative activity. Two distinct AgNOR counting methods have been previously shown to correlate with DNA ploidy and proliferative activity, respectively: the mean AgNOR count (mAgNOR) correlates more with ploidy, and the percentage of nuclei with > or = 5 AgNORs/nucleus (pAgNOR) reflects proliferative activity. We studied bone marrow specimens from 32 patients with B-CLL using both anti-IL-2R on the marrow aspirates and the AgNOR silver stain on marrow biopsy specimens, applying both AgNOR counts. All tumors were CD5+, CD19+, and CD19/CD20+. Sixteen tumors were IL-2R- (< 20% IL-2+ B cells), and 16 were IL-2R+ (> or = 20% IL-2R+ B-cells). No significant difference in morphology of bone marrow involvement was noted in the two groups. A male predominance was noted in the IL-2R+ group of patients (3:1). There was also a preponderance of lambda light chain expression in the IL-2R+ tumors (11/16) compared with the IL-2R- cases (5/16). Except for two cases, all tumors had mAgNOR counts within the diploid range (< 2.4). The 16 IL-2R- tumors had pAgNOR in the range of 0% to 7% (mean, 2.31 +/- 2.18 standard deviation), whereas the IL-2R+ tumors had pAgNOR ranging from 6% to 15% (mean, 10.20 +/- 2.70 standard deviation; P < .0001). This finding suggests that IL-2R+ B-CLL might represent a subgroup of tumors with higher proliferative activity.
In this paper we describe a new primer pair for the short tandem repeat (STR) D12S391 which makes it possible to obtain considerably shorter amplification fragments (125-173 bp), compared to the previously published primers (205-253 bp). The primers were tested on 70 samples with known genotypes, and no differing results were found. In sensitivity studies, forensic casework samples and DNA quality studies, we proved that the new primers can improve the efficiency of the amplification. Moreover, the resolution of this locus on denaturing PAGE followed by silver staining was dramatically improved. This improvement was found to be most valuable for typing the rare.3 variants known for this locus. We also present and propose a new method for silver staining denaturing acrylamide gels.
The numbers of silver-stained nucleolar organizer regions (AgNORs) were counted in uterine myomatous tumors, and compared with those in corresponding normal myometria. The mean number of AgNORs in myomatous tumors tended to increase according to the neoplastic changes. The mean number of AgNORs in leiomyosarcoma (6.4 +/- 0.9) was significantly higher than that in cellular leiomyoma (4.5 +/- 0.7, P < 0.01). or leiomyoma (3.0 +/- 0.5, P < 0.001). In the normal myometria, the mean number of AgNORs (3.3 +/- 0.4) in the premenopausal women showed a higher tendency than that in the post-menopausal women (2.4 +/- 0.5). These results therefore suggest that AgNOR counts may be useful for diagnosis of cellular activity in uterine myomatous tumors.
The present paper discusses the relevance of silver-stained Nucleolar Organizer Regions (AgNOR) to tumour pathology. First, the structural and functional aspects of AgNORs and the biological meaning of their quantitative variations have been reviewed to clearly define which indications can be obtained by the use of AgNOR. In continuously proliferating cells, AgNOR indicates the extent of ribosomal biogenesis, which is strictly related to the rapidity of cell proliferation. Therefore, AgNOR can be considered to represent a marker of cell proliferation rate and, in tumour pathology, should be used only for prognostic purposes. The predictive value of AgNOR, the only parameter which indicates the cell growth rate in situ in routinely processed cyto-histological samples, is strengthened by the combination with markers of cell growth kinetics (e.g. Ki67/MIB1).
A direct immunogold-silver staining (D-IGSS) method with 5nm colloidal gold-labeled rabbit anti-HFRS IgG antibodies was employed to detect HFRS virus antigen in the cytoplasm of white blood cells from HFRS patients and compared with indirect immunofluorescence assay (I-IFA). The results showed as follows: 1. The positive rate (87.5%) of HFRSV antigen detected by D-IGSS was significantly higher than that (65.6%) detected by I-IFA. in the white blood cells (P<0.05); 2. With the D-IGSS method the positive rates of HFRSV antigen in white blood cells of finger and vein blood were 81.3% and 87.5% respectively (P>0.05) with no significant difference but finger blood smears is more simple. We belive the D-IGSS method can be widely used for early diagnosis of HFRS at basic-level hospitals.
A silver staining method has been developed to study polymorphic proteins in bloodstains after isoelectric focusing. This method is highly sensitive and permits the detection of polymorphic proteins (i.e. alpha 1-antitrypsin, Gc and Tf C subtypes) in bloodstains as small as 0.2 microliter or less. The method is simple and reproducible and can be used after immunofixation. Blood stains can be identified after longer storage periods than is possible by using conventional staining methods.
Silver-stained nucleoli of rat hepatocytes were studied in norm, in liver cirrhosis produced by CCl4 poisoning and after cessation of the poisoning. Morphometric parameters of nucleoli were measured using a Videotest computer image analyser. Under cirrhosis the mean number of nucleoli per nucleus was determined to exceed their normal number by 1.27 times. The total volume of nucleoli in the nucleus also exceeded the normal level (by 1.15 times). 3 months after the end of CCl4-poisoning, these parameters decreased almost to normal values. A statistically significant correlation was revealed between the number of nucleoli and their total volume (0.881). Changes of the parameters also correlated with the total protein content in the hepatocytes. Possible reasons for this correlation are discussed. The ratio of the number of chromosomal NORs to the mean number of nucleoli in the nucleus is proposed to be used as a feature for comparative analysis of functional status of nucleoli in the nuclei of different ploidy and in cells of different animal species.
Silver staining of argyrophilic proteins of nucleolar organizer regions (Ag-NOR) has been used to evaluate a hyperactive state of cells. We studied the effects of sex steroids on the Ag-NOR proteins in the rabbit uterus. Estradiol-17beta (E2) caused uterine hypertrophy and increased the number of Ag-NOR dots in epithelial and stromal cells of the endometrium and smooth muscles cells in a dose dependent manner without proliferative change in the endometrium. Progestins including progesterone, medroxyprogesterone acetate (MPA), and norethindrone following E2 caused progestational proliferation of the endometrium with an increase in the Ag-NOR number to various extents, whereas methyltestosterone, a synthetic androgen, caused only a minimal proliferative change in the endometrium without an increase in the Ag-NOR number. Western blot analysis revealed that progesterone and MPA increased the amounts of a 100 kDa protein (nucleolin) and a 37 kDa protein (B23 protein) in the endometium and a marked proliferative change, whereas E2 did not. These results suggest that although the Ag-NOR number was enhanced by both progestin and estrogen, only progestins substantially increase the Ag-NOR protein in the rabbit uterus. This implies that an increase in the amount of nucleolin and B23 protein is associated with the proliferative potential of the cells.
Lactobacillus rhamnosus is often used in milk products because of its technological properties, however it is supposed to be related to human infections as well. Therefore, 23 L. rhamnosus strains, including the type strain, were investigated. Nine strains resulted from biotechnological sources, 14 isolates had a clinical background, 13 came from a swedish culture collection. Biochemical and physiological properties were proved by classical tests. Analysis of the total soluble cytoplasmatic protein patterns was performed with diamine silver staining, a technique not previously applied to lactobacilli. Classical tests were able to confirm all strains as L. rhamnosus. Analysis of protein patterns allowed to differentiate between three clusters. Cluster I contained the type strain and biotechnologically used strains and one clinical strain isolated in Berlin. The second consisted of both, technological and clinical strains. The third cluster contained clinical isolates alone. SDS-PAGE of proteins together with diamine silver staining seems to be helpful to detect intra-species differences. It was stated, that technologically used strains of L. rhamnosus could clearly be differentiated from each other and also from clinical isolates.
Some recently developed silver staining methods allow selective staining of acidic nucleolar proteins. Pretreating deparaffinized sections with Schiff's reagent improves the specificity of Goodpasture and Bloom's AgNOR staining (as modified by Kodama et al.) after aldehyde fixation.
Nineteen scorers from seven Cuban laboratories participated in this slide exercise designed to test the influence of the scorer on the accuracy, sensitivity and variability of the comet assay when a visual method of DNA damage evaluation is used. The assay was performed using human lymphocytes from a single donor exposed in vitro for 5 min at 0 degrees C to doses of 0, 5, 10, 25, 50, 100 and 200 microM of hydrogen peroxide. Each participant scored the same set of 14 coded slides with silver stained comets. The comets were classified visually into five categories according to the appearance resulting from the relative proportion of DNA in the tail. The extent of DNA damage was expressed in arbitrary units. At zero dose the median values of 12 scorers out of 19 were included between the values of the overall 25 and 75 per thousand. This proportion remains practically the same as the dose increases. The lowest dose detected by this method for the majority of scorers (11) was 10 microM. The coefficient of variation at the control dose was the highest (median value 26%), progressively declined to 20%, and starting from 25 microM, values are around 10%. The results of the exercise show the reliability of the silver staining and visual scoring for the comet method.
Immunogold-silver staining (IGSS) utilizes a histochemical method called autometallography (AMG) to amplify tiny gold particles to sizes easily visible both in light and electron microscopy. In both applications it is advisable to use the smallest possible gold diameters (1-6 nm) to obtain the highest sensitivity, thus, allowing minute amounts of the target substance to be demonstrated. Gold labels smaller than 10 nm in diameter have been clearly shown to give the highest labeling densities of antigen-antibody binding sites. AMG can be used for the detection of catalytic crystal lattices of metallic gold and silver, and sulfides or selenides of mercury, silver, copper, bismuth, and zinc. The method has its roots in "physical development" technique, transplanted from photography to histology by Liesegang at the beginning of this century. In 1981, a series of papers were published by one of us with the purpose of introducing a reliable and easy-to-handle technique for light microscopical and ultrastructural studies. AMG has a multitude of applications apart from its use in detecting tissue metals. These include the highly sensitive and efficient in situ colloidal gold tracing of peptides, proteins, and amines by immunocytochemistry using the IGSS method, of carbohydrates by lectin IGSS, and of nucleic acids by IGSS in situ hybridization, IGSS in situ polymerase chain reaction, and IGSS in situ self-sustained sequence replication-based amplification (in situ 3SR) techniques, the last two even performing with single-copy sensitivity. Applications of pre- and postembedding AMG for semithin and ultrathin tissue sections are described.
OBJECTIVE: To identify genes associated with the chronic progression of renal disease and a stragalus and angelica (A&A)'s renal protective effects. METHODS: The technique of silver staining mRNA differential display (DD) was used to investigate changes of gene expression in normal, sclerotic and A&A treated sclerotic kidneys. We isolated genes differentially expressed during the progression of renal disease which could be normalized by A&A. RESULTS: Several genes related to A&A's protective effects were isolated and one of them was confirmed by Northern blot. CONCLUSION: Silver staining mRNA differential display is a simple and effective technique for isolating differentially expressed genes. The isolated new gene may be related to the progression of chronic renal disease and contribute to A&A's protective effects.
A modified method of silver staining for native and denatured eucaryotic DNA in 1% agarose gel is described. This method is at least fivefold more sensitive than ethidium bromide staining, with a detection limit of 2.5 ng for total DNA. The calibration curve is linear within the range 5-30 ng of single-stranded and double-stranded DNA. This method is especially advantageous for electrophoretic assessment of DNA molecular weights.