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PubMed1976

Negative silver staining in A-T and satellite DNA-rich regions of human chromosomes.

Human metaphase chromosomes were stained with silver following a pretreatment with a heated alkaline solution. The most conspicuous feature of the stained metaphases was the omission of silver staining in the secondary constrictions of chromosomes 1,9 and 16, and on the distal Yq. Our evidence indicates that the negative silver binding is due to the preferential removal or alteration of non-histone proteins associated with these regions. The cytochemical significance of these findings is discussed.

Adenine Nucleotides↗
PubMed1992

Procedures for specific detection of silver-stained nucleolar proteins on western blots.

Nucleolar organizer region (NOR)-silver staining of the chromosomes and nucleoli is a method that enables the detection of proteins associated with the ribosomal genes. We adapted the most commonly used cytochemical NOR-silver staining techniques to Western-blotted proteins of HeLa cells, mimicking the silver staining of cells in situ, and testing several parameters that may influence the in situ reaction. Two of these techniques, both one-step methods with colloidal developers, were standardized to obtain reproducible results. The specificity of NOR staining is documented by: (a) only a few bands are revealed among the many proteins detected by total proteins staining on gels or blots; two major groups of bands are found around 100 KD and 40 KD that could correspond at least in part to nucleolin and B23 nucleolar proteins; (b) the silver staining of bands was not the result of the high relative protein concentrations; and (c) the same number of NOR-silver-stained bands was observed across a large range of protein concentrations. The reaction appeared to be specific for a subset of nucleolar proteins, because the same bands were observed with the use of nucleolar, nuclear, or total cell protein extracts, and the silver grains observed in electron microscopy were clearly confined to the nucleolar fibrillar centers and dense fibrillar component. The efficiency of the reaction was not modified by any of the tested fixative pre-treatments except that involving methanol. The presented standardization of NOR-silver staining on Western blots allows the characterization of the Ag-NOR proteins and their specific regions responsible for silver staining of the nucleolus.

Blotting, Western↗
PubMed1977

Visualization of ribosomal gene activity: silver stains proteins associated with rRNA transcribed from oocyte chromosomes.

Cricket oocyte chromosomes were stained with silver at pachytene when certain chromosome regions are active in rDNA amplification and rRNA transcription. The silver preferentially stained the known locations of 18S + 28S ribosomal cistrons. Cytochemical tests revealed that the silver binds neither to the rDNA nor transcribed rRNA, but rather to proteins which rapidly associate with the freshly-transcribed rRNA. As rRNA transcription proceeds, the quantity of silver stainable proteins progressively increases. The silver procedure can be used to visualize gene activity at the rDNA sites with conventional light microscopy.

Animals↗
PubMed1992

Detection of Pneumocystis carinii. Comparative study of monoclonal antibody and silver staining.

The sensitivity of Pneumocystis carinii detection using silver stain (Grocott method) was compared to that using the avidin-biotin-complex immunoperoxidase (IP) staining method with anti-P. carinii monoclonal antibody. Silver stain detected only cyst wall, whereas IP stained both cyst wall and trophozoites. Serial sections of formalin-fixed, paraffin-embedded autopsy lung specimens from 41 acquired immune deficiency syndrome patients in three disease categories were stained: I--premortem or autopsy diagnosis of P. carinii pneumonia (13 cases); II--history of treated P. carinii pneumonia but no P. carinii detected in autopsy tissue specimens (15 cases); and III--no clinical or autopsy evidence of P. carinii pneumonia (13 cases). Smears from 7 bronchoalveolar lavages (3 positive) and 11 induced sputa (1 positive) also were stained. All cases of P. carinii in category I were detected with equal sensitivity. P. carinii undetected by silver stain in category II and III cases and in bronchoalveolar lavages and induced sputa were not revealed by IP. Detection of trophozoites by IP did not improve sensitivity because the staining pattern was amorphous or focally granular, and thus easily confused with nonspecific staining of mucin or intracellular or free particulate material. Reliable identification of trophozoites could be made only with coexisting cyst structures. Silver staining was more advantageous because it also identified fungal infections and was faster and more cost effective than IP.

Antibodies, Monoclonal↗
PubMed1990

[Various activities of the nucleolus organizer region in normal and leukemic bone marrow cells: semiquantitative data and computer- assisted image analysis by silver staining].

A cytochemical technique with silver nitrate staining was used to study the nucleolar organizer activity in metaphase spread of bone marrow cells from 13 patients with acute lymphocytic leukemia (ALL), 11 patients with chronic myelogenous leukemia (CML), 7 patients with acute myelogenous leukemia (AML), and 4 normal persons. Additionally, computer-assisted image analysis was used to quantitate the amount of silver staining in interphase nuclei of bone marrow cells from 6 untreated ALL patients, 3 normal subjects and 1 bone-marrow-transplant recipient. The results obtained have indicated that the nucleolar organizer reactivity (NOR) is significantly lower in the control group than that in ALL patients. NOR activity level is significantly lower in both CML patients in chronic phase, and AML patients than in the ALL group, and is similar to that in the control group. When the data obtained for the interphase nuclei were compared with those obtained for the metaphase spread, a strong correlation was recorded between the fraction of bone-marrow metaphases stained positively with silver, the average number of silver-positive nucleolar organizer regions per metaphase, and the amount of silver staining in the interphase nuclei. Silver staining used for the detection of these disease-related differences in NOR activity can serve as a diagnostic procedure in evaluating human leukemias. The computer-assisted image analysis of bone marrow cell interphase nuclei would be useful for more accurate resolving biological and medical problems.

Bone Marrow↗
PubMed1999

Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: a method for the removal of silver ions to enhance sensitivity.

Mass spectrometry is a powerful technique for the identification of proteins at nanogram quantities. However, some degree of sample preparation prior to mass spectrometry is required, and silver-stained protein gel samples are most problematic. Here we report our strategy to obtain peptide mass profiles from silver-stained protein gel samples from one- or two-dimensional gels by destaining prior to enzymatic digestion. This study demonstrates that by using the destaining method, the sensitivity and quality of mass spectra is increased for matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometric analysis, permitting more proteins to be identified by peptide mass database analysis.

Acrylic Resins↗
PubMed2006

Time-based analysis of silver-stained proteins in acrylamide gels.

Silver staining of proteins after PAGE often remains the method of choice in many laboratories. Nevertheless, it is known that quantification of protein levels is keenly restricted to a small range of protein concentrations leading to an over- or underestimation of protein amounts. To overcome this, a time-based analysis method was developed to avoid the saturation effect of the silver-staining reaction, thus resulting in an improved dynamic range of the gel image produced and therefore better quantification of proteins. Instead of the well-known end-point image analysis, gray intensities of time series images of a developing gel are determined and times until a threshold gray value is reached are calculated. These times are used to calculate a new grayscale image which can be analyzed using commercial image processing software.

Animals↗
PubMed1979

Age-dependent variability of ribosomal RNA-gene activity in man as determined from frequencies of silver staining nucleolus organizing regions on metaphase chromosomes of lymphocytes and fibroblasts.

Frequencies of silver staining nucleolus organizing regions (NORs) have been determined in lymphocytes and fibroblasts from very young and from aged persons. Since silver staining of NORs is associated with activity of ribosomal RNA-genes, we used this approach to investigate a possible inactivation of these genes during aging. Our lymphocyte data are based on a study per age-group of 220 metaphases from 10 subjects. Although in both age-groups modal numbers of silver staining chromosomes per metaphase had similar ranges over the subjects, the frequency of metaphases containing the maximal number of staining chromosomes was in the old age-group (80--89 years) significantly lower than in the young age-group (less than 1 year old). In fibroblasts, of which 75 metaphases from 4 subjects were included per age-group, differences were more pronounced. Modal numbers of silver staining chromosomes were for the aged persons (69--83 years) lower than for the young children (less than 1 year old). Highly significant differences were observed between both groups in frequency of metaphases containing the maximal number of positively reacting acrocentric chromosomes and, more in general, in frequencies of silver staining D- and G-group chromosomes, the lower frequencies being found in the old age-group. We propose the term NOR-junctions as distinct from satellite associations for arrangements of acrocentric chromosomes which after silver staining are visibly connected at their NORs. The number of acrocentric chromosomes involved in lymphocyte NOR-junctions of aged people was significantly higher than the number of joined acrocentrics in young children. The frequency of these NOR-junctions themselves, irrespective of the number of chromsomes involved, was higher for aged persons than for young children, although this difference appeared to be statistically not significantly higher than in fibroblasts. Also based on qualitative observations from our study we discuss tcehnical and biological problems of our approach to study cell aging in vivo by means of silver staining of NORs. We conclude that in man, reflected by the difference in frequencies of silver staining NORs between young and aged persons, a rather extensive loss of ribosomal RNA-gene activity may occur during aging.

Aged↗
PubMed2004

About the mechanism of interference of silver staining with peptide mass spectrometry.

The mechanism by which silver staining of proteins in polyacrylamide gels interferes with mass spectrometry of peptides produced by proteolysis has been investigated. It was demonstrated that this interference increases with time between silver staining and gel processing, although the silver image is constant. This suggested an important role of the formaldehyde used in silver staining development in this interference process. Consequently, a formaldehyde-free staining protocol has been devised, using carbohydrazide as the developing agent. This protocol showed much increased peptide coverage and retained the sensitivity of silver staining. These results were however obtained at the expense of an increased background in the stained gels and of a reduced staining homogeneity.

Electrophoresis, Gel, Two-Dimensional↗
PubMed1983

Optimal detection of oligoclonal bands in CSF by silver stain.

We applied a sensitive silver nitrate stain to SDS-polyacrylamide gels to detect oligoclonal bands in unconcentrated CSF. Silver staining took less than 3 hours and gave easily readable bands in 93% of MS patients. In contrast, standard Coomassie Blue stain was positive in only 53%. Maximum resolution and numbers of bands resulted when silver staining was used on 10 to 20 microliters of CSF. Silver staining of SDS-polyacrylamide gels permits reliable oligoclonal band detection on small volumes of unconcentrated CSF.

Electrophoresis, Polyacrylamide Gel↗
PubMed1984

Further evidence that phosphoprotein C23 (110 kD/pI 5.1) is the nucleolar silver staining protein.

Previously we demonstrated a similar distribution between nucleolar organizing region-(NOR)-specific silver staining and localization of nucleolar phosphoprotein C23 (MW 110 kD/pI 5.1) [1, 2]. We now report that under fixation conditions which allow for antibody binding and subsequent silver staining, monoclonal antibody against protein C23 blocks NOR silver staining as well as silver staining in interphase nucleoli. Monoclonal antibody against nucleolar phosphoprotein B23 (MW 37 kD/pI 5.1) did not block silver staining in either NORs or interphase nucleoli. These, along with earlier observations, provide evidence that nucleolar phosphoprotein C23 is the major silver staining protein of the nucleolus and that it is directly or indirectly associated with rDNA.

Animals↗
PubMed1989

Effects of age, sex and multiple endocrine neoplasia type-II on silver stained nucleolar organizer regions.

Silver stained nucleolar organizer regions (AgNORs) were studied in phytohemagglutinin (PHA)-stimulated lymphocytes from 55 Caucasian control individuals (34 females with average age of 24 years and age range 19 weeks gestation to 87 years; 21 males with average age of 31 years and age range 29 weeks gestation to 72 years) and 13 individuals (7 females, 6 males; average age 38.8 years with age range 25-58 years) with multiple endocrine neoplasia-type II (MEN-II), an autosomal dominant malignancy with increased chromosome breakage. For the first time, AgNORs were examined in lymphocytes from normal fetuses and patients with MEN-II in order to determine the effects of age, sex or malignancy on the number of AgNORs. No significant difference in the average number of AgNORs were found in fetal cells (8.2 +/- S.D. 0.7/cell) when compared with cells from older individuals including those over 65 years of age (8.0 +/- S.D. 0.8/cell). There was a statistically significant negative correlation (P less than 0.05) between the modal number of AgNORs on G but not D chromosomes in both males and females. A negative correlation was also found between the mean number of AgNORs and age but was not statistically significant. The average number of AgNORs in the MEN-II individuals was 8.5 +/- S.D. 0.7/cell, which was not significantly different than 8.2 +/- S.D. 0.7/cell observed in age-matched control subjects.

Adolescent↗
PubMed1985

Detection of host-derived contaminants in products of recombinant DNA technology in E. coli: a comparison of silver-staining and immunoblotting.

Contamination of medicines produced in E. coli by recombinant DNA methodology with host-cell proteins is considered a potential problem with this type of method. In this report techniques for the detection of trace quantities of host-cell proteins in SDS-gel electrophoretograms were examined. Detection of E. coli proteins by immunoblotting, using antisera raised in rabbits to lysates of E. coli, was compared with detection using the ultrasensitive silver stain. Silver staining detected a larger number of E. coli proteins in a one-dimensional electrophoresis system than did immunoblotting. Proteins that were markedly antigenic in the rabbit were detected at a greater sensitivity by the immunoblotting approach. Both techniques detected contaminant proteins in a preparation of methionyl human growth hormone produced in E. coli known to be contaminated with host-cell proteins. No contaminating proteins were seen by either technique in more rigorously purified preparations of growth hormone. A combination of these two approaches would provide useful evidence of purity of medicines produced by recombinant DNA technology, and is potentially applicable to a wide range of host-vector systems.

Animals↗
PubMed1987

A comparative study of a silver stain and monoclonal antibody reactions on Alzheimer's neurofibrillary tangles.

Serial sections of brains of eight cases with Alzheimer's disease were stained by methods utilising silver and three different monoclonal antibodies. The numbers of Alzheimer's neurofibrillary tangles (NFT) were counted in the silver stained sections and compared with the numbers detected by three monoclonal anti-NF antibodies: RT97, BF10 and 8D8. The monoclonal antibody (Mab) BF10 demonstrated NFTs more clearly than Mab 8D8, but neither detected as many tangles as the silver staining method. Mab RT97 reacted well with NFTs in freshly processed specimens but not following prolonged fixation.

Aged↗
PubMed1990

An improved silver staining technique as an alternative nuclear or combined nuclear nerve-fiber impregnation for comparative light-, secondary and backscattered electron scanning microscopy.

Slices of rat brain were stained by a new silver impregnation technique. This method takes into consideration the pH-dependent differences of silver stain affinity of nerve tissues and can be used alternatively as a stain for nuclei or as a method for combined demonstration of nuclei nerves fibers. The slices were studied at the light microscopical (LM) level and subsequently with a scanning electron microscope, using secondary (SSEM = classical SEM), and backscattered electron detectors (BSEM). This new silver staining technique offers the opportunity of comparative studies with regard to different information acquired with LM, SSEM and BSEM. The described method allows to distinguish between nervous and glial tissue without necessarily damaging the glial tissue surrounding the nerve fibers. Specifically, scanning electron microscopy with backscattered electron detector of in situ preparations provides a higher contrast of stained and unstained tissue and increased depth of focus as compared to secondary electron detectors.

Animals↗
PubMed1994

Enhanced staining of bacterial flagella using aged mordant in the silver stain.

Intensity of bacterial flagella staining using a modified silver stain was increased by aging the mordant for one week at room temperature. The use of aged mordant increased the apparent diameters of stained flagella and resulted in a darker stain. The mordant remained stable for at least four months at room temperature. The staining protocol presented allows application to liquid or solid cultures.

Bacillus subtilis↗