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Use of tannic acid and silver enhancer to improve staining for electron microscopy and immunogold labeling.

Staining by uranyl acetate and lead citrate (UA-LC) of immunolabeled sections of unfixed glomerular basement membrane (GBM) digests embedded in low-acid glycol methacrylate (LA-GMA) is poor. The following were investigated for their ability to enhance contrast when applied to sections before UA-LC: potassium permanganate, phosphotungstic acid, gold chloride, osmium tetroxide, glutaraldehyde-osmium tetroxide, colloidal gold-silver enhancer, tannic acid, and glutaraldehyde-tannic acid. Silver enhancer (2 min incubation, no sodium thiosulfate step) gave dense GBM staining but not with immunogold labeling. Silver enhancer is recommended as a simple alternative to routine silver stains but not for contrast enhancement with immunogold labeling. Tannic acid (1% for 1 min or 0.005% for 10 sec) and glutaraldehyde (2.5% for 5 min) followed by tannic acid enhanced contrast and, when applied after immunolabeling, did not appear to affect probe levels. Tannic acid also enhanced the staining of fixed tissue in LR Gold acrylic resin and LA-GMA, but not of glutaraldehyde-osmium-fixed tissue in epoxy resin, even after sodium metaperiodate treatment. Treatment of sections with tannic acid is recommended for contrast enhancement in immunogold studies when osmium and tannic acid post-fixation must be avoided and the tissue is embedded in methacrylate or acrylic resin.

Basement Membrane↗

Optimal visualization of immunogold-silver staining of hepatic PEPCK with epipolarized light microscopy.

We used immunogold-silver staining to localize phosphoenolpyruvate carboxykinase in 10 microns cryosections of 4% paraformaldehyde perfusion-fixed normal male rat liver. The resolution and sensitivity of detection were improved by epipolarized light microscopy of 0.5 microns semi-thin plastic sections prepared from these pre-embedding immunogold-silver-enhanced 10-microns thick cryosections. Epipolarized light combined with transmitted light simultaneously demonstrated antigenic sites (visualized with epipolarized light illumination) and tissue morphology (revealed by transmitted light). To optimize the conditions for high resolution, an oil immersion objective lens (x 100) with adjustable iris diaphragm was used with different intensity settings for both light sources. Our observations indicate that if the intensity of the transmitted light is too high, the visibility of the gold-cored silver grains by epipolarized illumination is decreased; if the intensity of epipolarized light is too strong, haloes appear around the gold-cored silver particles. By adjusting the aperture in the objective lens and the neutral density filter in the transmitted light pathway to balance the intensities of transmitted and epipolarized light, an optimal image is obtained that shows the maximal number of antigenic sites and excellent morphology.

Animals↗

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↗

[Effects of metals (silver, nickel, cobalt and chromium) on the reactive oxygen species generating capacity of human neutrophils and on the serum opsonic activity].

Many metal materials are used in clinical medicine and the number used is increasing. However, there are a few papers concerning the biological effects, especially effects of clinically used metals on cellular immunity. In this paper, silver (Ag), nickel (Ni), cobalt (Co), and chromium (Cr), clinically commonly used metals, the effects of which on neutrophilic activity have already reported using the luminol-dependent chemiluminescence (LDCL) method, were examined to determine their influence on the reactive oxygen species (ROS) generating capacity of human neutrophils and on a serum opsonic activity. Silver suppressed the ROS generating capacity of neutrophils in a dose-dependent manner but did not affect the serum opsonic activity. Nickel also suppressed the ROS generating capacity of neutrophils but, at some concentrations, caused the serum opsonic activity to increase. In contrast, Cobalt increased both the ROS generating capacity of neutrophils and the serum opsonic activity. Like silver, chromium did not affect the serum opsonic activity and suppressed the ROS generating capacity of neutrophils. However, the suppression of ROS generating by chromium occurred differently from that of silver. In the cases of nickel and cobalt, effects of these metals on hemolyzing activity of serum complements (CH-50) were measured and consumption of complements was observed at the same metal concentration at which serum opsonic activity was elevated. In this paper, we report that some metals influence not only the ROS generating capacity of neutrophils but also the serum opsonic activity, results thought to be important for more details studies of the effects of metals on cellular immunity.

Cells, Cultured↗

Control of Pythium root rot on hydroponically grown cucumbers with silver-coated cloth.

Silver-coated cloth (SCC) effectively controlled root rot that was caused by Pythium aphanidermatum in hydroponically grown cucumber plants. The presence of SCC in the hydroponic solution reduced the root rot from 100% to 10% 20 days after inoculation with zoospores of P. aphanidermatum. It was suggested that the inhibition of SCC was caused not only by the silver ion dissolved from SCC, but also by the metallic silver and silver compounds formed on the surface of the root.

Cucumis sativus↗

Adsorption of S-S containing proteins on a colloidal silver surface studied by surface-enhanced Raman spectroscopy.

We present a Raman and surface-enhanced Raman scattering (SERS) study of the following proteins containing S-S group(s): alpha chymotrypsin (alpha-CHT), insulin, lysozyme, oxytocin (OXT), Streptomyces subtilisin inhibitor (SSI), and trypsin inhibitor (STI). The SERS study is performed in order to understand the adsorption mechanism of the above-mentioned proteins on a colloidal silver surface. The SERS spectra presented here show bands associated mainly with aromatic amino acid vibrations. In addition, two distinct vibrations of the -C-S-S-C- fragment are observed in the Raman and SERS spectra, i.e., nu(SS) and nu(CS). The enhancement of the nu(SS) vibration in the SERS spectra yields evidence that the intact disulfide bridge(s) is (are) located near the silver surface. This finding is supported by the presence of the nu(CS) mode(s). The presence of nus(COO-) and nu(C-COO-) in the SERS spectra in the 1384-1399 cm(-1) and 909-939 cm(-1) regions, respectively, indicate that the negatively charged COO- groups (aspartic and glutamic acids) assist in the binding on the positively charged silver surface. The Raman amide I and III bands observed in the 1621-1633 and 1261-1289 cm(-1) ranges, respectively, indicate that the alpha-helical conformation is favored for binding to the surface over the random coil or beta-sheet conformations. In addition, the presence of the imino group of Trp and/or His indicates that these amino acid residues may also bind to the silver sol.

Adsorption↗

Surface-enhanced Raman spectroscopy using silver-coated porous glass-ceramic substrates.

Surface-enhanced Raman scattering (SERS) has been studied using a silver-coated porous glass-ceramic material as a new type of substrate. The porous glass-ceramic is in the CaO-TiO2-P2O5 system prepared by controlled crystallization and subsequent chemical leaching of the dense glass-ceramic, leaving a solid skeleton with pores ranging in size from 50 nm to submicrometer. Silver was coated on the surface of the porous glass-ceramic by radio frequency (RF) sputtering or e-beam evaporation in vacuum. SERS spectra of excellent quality were obtained from several dyes and carboxylic acid molecules, including rhodamine 6G, crystal violet, isonicotinic acid, and benzoic acid, using this new substrate. This new substrate showed a good compatibility with these molecules. The porous glass ceramic with a nanometer-structured surface accommodated both test molecules and silver film. The absorbed molecules were therefore better interfaced with silver for surface-enhanced Raman scattering.

Benzoic Acid↗

Efficacy of subcutaneous silver-impregnated cuffs in preventing central venous catheter infections.

STUDY OBJECTIVES: To determine the efficacy of an attachable subcutaneous silver-impregnated cuff in preventing local central venous catheter (CVC)-related infection and catheter-related sepsis in critically ill surgical patients. DESIGN: A prospective analysis of the use of an attachable subcutaneous silver-impregnated cuff compared with a control group in two consecutive time periods. SETTING: Two surgical ICUs at the Queen's Medical Center at the University of Hawaii Surgical Residency Program, Honolulu. PATIENTS: All surgical ICU patients requiring insertion of central catheters. INTERVENTIONS: None. MEASUREMENT AND MAIN RESULT: Two hundred thirty-five CVCs in 154 patients were prospectively evaluated. Silver-impregnated cuffs were used in the first 100 catheters, but none were used in the remaining 135 catheters. The incidence of catheter-related infection in both groups was 15% and 20%, respectively, not statistically significant. Catheter-related sepsis was 3% in both groups. CONCLUSIONS: The use of an attachable subcutaneous silver-impregnated cuff failed to decrease the incidence of CVC-related infection and sepsis.

APACHE↗

Silver acetate gum as a deterrent to smoking.

Silver acetate chewing gum, a nonprescription medication, produces an unpleasant metallic taste in the mouth of individuals who consume tobacco products in conjunction with this smoking deterrent. Use of the product leads to self-induced aversive conditioning. In the present double-blind controlled study, subjects using silver acetate for three weeks of treatment had a smoking cessation rate of 15 out of 136 (11 percent, p = 0.02). Placebo subjects had a smoking cessation rate of 6 out of 146 (4 percent, p = .102). Without further treatment, the group using silver acetate demonstrated a 7 percent nonsmoking rate at four months compared with a 3 percent nonsmoking rate for the placebo group. Silver acetate demonstrated a modest benefit over placebo as a smoking deterrent in a minimal intervention and highly cost-effective treatment setting.

Acetates↗

Perineuronal nets of proteoglycans in the adult mouse brain, with special reference to their reactions to Gömöri's ammoniacal silver and Ehrlich's methylene blue.

As our previous studies have indicated, many subsets of neurons in the vertebrate brain possess a sulfated proteoglycan surface coat which reacts to cationic iron colloid and aldehyde fuchsin. The present study demonstrated that this surface coat is supravitally stained with Ehrlich's methylene blue, and doubly with this blue and aldehyde fuchsin, a finding suggesting its being identical to Cajal's superficial reticulum (red superficial) and to Golgi's reticular coating (revetement reticulare). The perineuronal surface coat was further stained with Gömöri's ammoniacal silver, and doubly with this silver and cationic iron colloid. These neurons with such a proteoglycan surface coat usually expressed cell surface glycoproteins which were labeled with lectin Wisteria floribunda agglutinin. Hyaluronidase digestion did not interfere with this lectin labeling of the glycoproteins, methylene blue and Gömöri's ammoniacal silver staining of the surface coat, while it erased the cationic iron colloid and aldehyde fuchsin staining of the surface coat. These findings suggest that the perineuronal proteoglycan surface coat is associated with some additional molecules which are resistant to hyaluronidase digestion and stainable with methylene blue and Gömöri's ammoniacal silver. The possibility is suggested that these molecules might represent "ligand proteoglycans" connecting the perineuronal proteoglycans and cell surface glycoproteins.

Animals↗

Influence of dissolved organic matter source on silver toxicity to Pimephales promelas.

In the environment, the formation of organic and inorganic silver complexes can decrease Ag bioavailability (toxicity) to aquatic organisms. However, current water quality regulations do not consider the protective effects of water quality parameters such as dissolved organic carbon (DOC) concentration. To determine the effect of DOC concentration and source on silver toxicity, nine different natural organic matter isolates were used in 96-h static-renewal toxicity tests with fathead minnow (Pimephales promelas). The 96-h dissolved silver median lethal concentrations (LC50) among different sources of dissolved organic matter varied by up to fivefold (4.5-23.3 microg/L). Further, toxicity tests with organic matter from the site with the lowest 96-h LC50 value suggested only limited additional attenuation of silver toxicity when DOC concentration was increased from 5.1 to 14.0 mg/L. With this site excluded, we found little more than a twofold difference among 96-h dissolved Ag LC50s for the remaining sources (10.1-23.3 microg/L). However, significant toxicological differences among sites remained. It was apparent that organic matter from different sources varied both chemically and toxicologically, but no conclusions could be drawn that related compositional variation to observed Ag toxicity for these isolates.

Animals↗

Solid phase extraction and spectrophotometric determination of silver with 2-(2-quinolylazo)-5-diethylaminophenol as chromogenic reagent.

A new chromogenic reagent, 2-(2-quinolylazo)-5-diethylaminophenol (QADEAP), was synthesized. A sensitive, selective and rapid method has been developed for the determination of microg/L level silver ion based on the rapid reaction of silver(l) with QADEAP and the solid phase extraction of the colored chelate with C18 cartridge. The QADEAP reacts with Ag(l) to form a violet chelate of a molar ratio 1:2 (silver to QADEAP) in pH 3.5-8.0. This chelate was prconcentrated by solid phase extraction with C18 cartridge. An enrichment factor of 100 was achieved. The molar absorptivity of the chelate is 1.30 x 10(5) L mol(-1) cm(-1) at 590 nm in measured solution. Beer's law is obeyed in the range of 0.01-0.6 microg/ml. The relative standard deviation for eleven replicate samples of 0.01 microg/ml is 1.15%. The detection limit is 0.02 microg/L in the original samples. This method was applied to the determination of microg/L level silver ion in water with good results.

Aminophenols↗

Accumulation of copper and silver onto cell body and its effect on the inactivation of Pseudomonas aeruginosa.

Pseudomonas aeruginosa, a gram-negative rod bacterium, is a causative agent of waterborne pneumonia and presents high tolerance against conventional disinfectants. The inorganic biocidal reagents, copper and silver, were applied to inactivate P. aeruginosa inoculated in a synthetic drinking water (SDW). Additionally, the relationship of the specific amount of accumulated copper and silver reagents (Cs) on P. aeruginosa with inactivation profile was elucidated in this study. Flow cytometry (FCM) following staining with SYTO 9 and PI was used for detection of bacterial viability and density. Individual copper and silver reagents, and their combination, exhibited excellent biocidal abilities even at the concentration of 0.05 mgCu/L and 0.005 mgAg/L. The critical amounts of accumulated disinfectant (Cs) were calculated at 2.82 x 10(-7) microgCu/cells and 5.13 x 10(-8) microgAg/cells; at an incubation of 70 h. Consequently, the role of disinfectant on the inactivation of P. aeruginosa and the assessment of biocidal ability of copper, silver, and their combination were successfully explained by evaluating the terms Cs and Cc.

Copper↗

A silver carbonate method for cell counts of neurons and glial elements on paraffin embedded brain tissue.

A modification of the Del Rio-Hortega method for the demonstration of central nervous system elements is presented. This silver impregnation technique is particularly useful for the classification of cell types for quantitative differential cell counts. Formalin fixed paraffin sections are immersed in formol-ammonium bromide for 1 1/2 hours; this solution is an excellent mordant for various silver nitrate stains. The samples are stained for 20 to 60 minutes in a silver carbonate solution (25 ml of 25% silver nitrate combined with 200 ml of 5% sodium carbonate) and then reduced in a 1% formaldehyde solution to which 20 drops of acetic acid have been added. Finally, the slides are fixed in sodium thiosulfate, rinsed in tap water, dehydrated, cleared, and mounted. This procedure will enable this investigator to identify neurons, oligodendroglia, and astrocytes on the basis of their nuclear staining as well as to demonstrate the laminae of brain tissue since the method allows differentiation of cell layers and fiber tracts.

Animals↗

A silver impregnation technique for the study of steroid receptors in central nervous system tissue prepared for autoradiography.

The commonly used silver stains were found to be unsatisfactory for nervous tissue processed for autoradiography. A silver impregnation procedure for central nervous system tissues prepared for the autoradiographic study of steroid receptors is described. The procedure is a combination of several silver and reticular strains made up in solutions containing dimethylsulfoxide. The technique clearly distinguishes perikarya of neurons, brain nuclei and fiber tracts without substantial loss of silver grains, and thus greatly facilitates the identification of steroid receptor nuclei at all levels of the central nervous system.

Animals↗

A method for combined C-banding and silver staining.

A reliable technique for combined C-banding and silver staining of metaphase chromosomes which uses trypsinization is described. Slides are first immersed in dilute HCl to remove residual cytoplasm from around the chromosomes. They are then treated with saturated barium hydroxide and incubated overnight in saline sodium citrate (0.30 M NaCl, 0.03 M sodium citrate, adjusted to pH 7.0 with HCl). Following the C-banding pretreatment, a two-step method of silver staining which employs a protective colloidal developer is used to stain the nucleolar organizer regions (NORs) of the chromosomes. Silver staining is followed by trypsinization to remove extraneous silver precipitate from the chromosome arms which permits the C-bands to be stained with Giemsa. The method works equally well with fresh and aged mitotic chromosome preparations and gives consistent staining of both heterochromatin and active NORs in metaphases across the slide.

Chromosome Banding↗

A method for silver impregnation of mammary myoepithelia.

Histochemical methods for microscopic visualization of mammary myoepithelial cells all yielded considerable variation in completeness of myoepithelial cell staining. Although extremely variable, silver impregnation occasionally gave tissue sections containing myoepithelia having excellent microanatomical detail and contrast with other tissue elements. Consequently, sources of variation in the silver technique were considered. Composition of the tissue fixative and pH of the silver impregnating solution were most critical. A final method is presented which gives consistent, complete silver impregnation of myoepithelia, where both the cell body and cell processes are clearly evident. The staining procedure is not light sensitive, nor is acid cleaning of glassware necessary. Tissue sections from lactating mouse, rat, hamster and goat are presented; tissue from other species should stain as well. The procedure should greatly facilitate the study of the function of myoepithelial cells and the visualization of these cells in mammary pathology.

Animals↗

Silver staining in clinical cytogenetics.

Silver staining of human chromosomes at prometaphase or metaphase identifies variants in the stalk (nucleolar organizing) regions of acrocentric chromosomes (Nos. 13, 14, 15, 21, 22). Variants are defined by size, number, and morphology of silver staining areas. They are heritable polymorphisms and have not been associated with clinical abnormalities. However, these variants are useful in clinical cytogenetics, specifically in studies attempting to determine whether genetic material has been gained or lost in chromosomal rearrangements, the origin of chromosomal aberrations, the origin of cells in tissue culture, the chromosomal location of single genes, clonal origin of tumors, the zygosity of twins, and paternity. Some chromosomal aberrations require silver staining for their definition. Because loss of the stalk regions per se is apparently not deleterious, demonstration that chromosomal breaks occurred within this region without concomitant loss or gain of genetic material essential for normal human development provides basis for a good prognosis for the individual with the chromosomal rearrangement resulting from such breakage. The principle underlying most of the other applications is to determine whether variants being compared are identical or dissimilar, and to make inferences from these results (e.g., variants in monozygotic twins should all be identical, whereas in dizygotic twins they are as similar as in any pair of sibs). Silver staining is a valuable technique for special questions in clinical analysis.

Chromosome Aberrations↗