Rapid Bielschowsky silver impregnation method using microwave heating.

The Bielschowsky silver impregnation method has been used extensively to demonstrate neuronal processes including dendrites, axons and neurofibrils. In this study, we examined the differences in the time required for and the staining quality of the Bielschowsky method for neuronal processes when microwave heating was used instead of processing at room temperature. For this purpose, a control group of sections stained according to the conventional method at room temperature was compared to an experimental group stained in a microwave oven at 180 W for 2, 4 and 1 min in 2% silver nitrate, ammoniacal silver nitrate and gold chloride, respectively. Light microscopic examination demonstrated that the normal structure was preserved in both groups and that there was no difference in the staining quality between the control and the microwave groups. In addition, staining time for this procedure was reduced to 8 min by using the microwave oven. Our study revealed that microwave irradiation can be used safely for Bielschowsky silver impregnation of neuronal tissues.     

Rapid Bielschowsky silver impregnation method using microwave heating

The Bielschowsky silver impregnation method has been used extensively to demonstrate neuronal processes including dendrites, axons and neurofibrils. In this study, we examined the differences in the time required for and the staining quality of the Bielschowsky method for neuronal processes when microwave heating was used instead of processing at room temperature. For this purpose, a control group of sections stained according to the conventional method at room temperature was compared to an experimental group stained in a microwave oven at 180 W for 2, 4 and 1 min in 2% silver nitrate, ammoniacal silver nitrate and gold chloride, respectively. Light microscopic examination demonstrated that the normal structure was preserved in both groups and that there was no difference in the staining quality between the control and the microwave groups. In addition, staining time for this procedure was reduced to 8 min by using the microwave oven. Our study revealed that microwave irradiation can be used safely for Bielschowsky silver impregnation of neuronal tissues.     

Rapid Bielschowsky silver impregnation method using microwave heating

The Bielschowsky silver impregnation method has been used extensively to demonstrate neuronal processes including dendrites, axons and neurofibrils. In this study, we examined the differences in the time required for and the staining quality of the Bielschowsky method for neuronal processes when microwave heating was used instead of processing at room temperature. For this purpose, a control group of sections stained according to the conventional method at room temperature was compared to an experimental group stained in a microwave oven at 180 W for 2, 4 and 1 min in 2% silver nitrate, ammoniacal silver nitrate and gold chloride, respectively. Light microscopic examination demonstrated that the normal structure was preserved in both groups and that there was no difference in the staining quality between the control and the microwave groups. In addition, staining time for this procedure was reduced to 8 min by using the microwave oven. Our study revealed that microwave irradiation can be used safely for Bielschowsky silver impregnation of neuronal tissues.     

Microwave-stimulated glutaraldehyde and osmium tetroxide fixation of plant tissue: ultrastructural preservation in seconds.

Microwave-enhanced fixation of animal tissues for electron microscopy has gained in interest in recent years. Attempts to use microwave irradiation for the preparation of plant tissues are rare. In this study; I report on microwave conditions which allow a high quality preservation of plant cell structure. Tissues used were: internodes of Chara vulgaris, leaves of Hordeum vulgare, root tips of Lepidium sativum. Microwave irradiation was done with a commercial microwave oven (Sharp R-5975). Fixatives used were: 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2 and 1% osmium tetroxide in veronal/acetate buffer, pH 7.2. Conventional fixations with glutaraldehyde/osmium were compared with microwave fixations. Examinations of thin sections showed that microwave fixation (glutaraldehyde or sequential aldehyde/osmium) is an attractive and rapid alternative method for processing plant tissues for electron microscopy. The optimal conditions found were: microwave oven at power level 50 W, 6.5 ml of fixative solution, irradiation times between 32-34 s, final temperature between 40 degrees C and 47 degrees C.     

Nucleolin is an Ag-NOR protein; this property is determined by its amino-terminal domain independently of its phosphorylation state.

The Ag-NOR proteins are defined as markers of "active" ribosomal genes. They correspond to a set of proteins specifically located in the nucleolar organizer regions (NORs), but have not yet been clearly identified. We adapted the specific detection method of the Ag-NOR proteins to Western blots in order to identify these proteins. Using a purified protein, Western blots, and immunological characterization, the present study brings the first direct evidence leading to the identity of one Ag-NOR protein. We found that nucleolin is specifically revealed by Ag-NOR staining. Using different nucleolin fragments generated by CNBr cleavage and by overexpression in Escherichia coli, we demonstrate that the amino-terminal domain of nucleolin and not the carboxy-part of the protein is involved in silver staining. Moreover, as the pattern of staining does not vary using casein kinase II- and cdc2-phosphorylated nucleolin or dephosphorylated nucleolin, we conclude that the reduction of the silver ions is not linked to the phosphorylation state of the molecule. We propose that the concentration of acidic amino acids in the amino-terminal domain of nucleolin is responsible for Ag-NOR staining. This hypothesis is also supported by the finding that poly L-glutamic acid peptides are silver stained. These results provide data that can be used to explain the specificity of Ag-NOR staining. Furthermore, we clearly establish that proteolysis of the amino-terminal Ag-NOR-sensitive part of nucleolin occurs in vitro, leading to the accumulation of the carboxy-terminal Ag-NOR-negative part of the protein. We argue that this cleavage occurs in vivo as already proposed, bearing in mind that nucleolin is present in the fibrillar and in the granular component of the nucleolus, whereas no Ag-NOR staining is observed in the latter nucleolar component.     

Silver Binding Nucleolar Organizer Regions In Adrenocortical Neoplasia

The possible contribution of the silver colloid technique for staining nucleolar organizer regions in the distinction between benign and malignant adrenocortical neoplasms was investigated. Nine cases of adenoma, eight cases of carcinoma, nine cases of hyperplasia and four normal adrenal cortex specimens were examined. The mean silver binding nucleolar organizer region (Ag-NOR) value in adenoma was 4.29, and in carcinoma 7.16 (P less than 0.001). Adenomas with diameters greater than 3 cm had significantly higher Ag-NOR counts than smaller adenomas. For normal cortex, the mean Ag-NOR value was 2.05 and in hyperplasia, 3.62. The results indicate that the Ag-NOR technique can help in differential diagnosis between benign and malignant adrenocortical lesions and thus may have a prognostic value.     

Simultaneous high resolution localization of Ag-NOR proteins and nucleoproteins in interphasic and mitotic nuclei

Summary Silver stainable proteins of the Nucleolar Organizer Regions (Ag-NOR proteins) of human breast cancer tissues have been localized at the electron microscopical level with a new method which combines a simple and reproducible one step Ag-NOR staining method combined with an acetylation procedure. This new method allows the fine identification of nucleolar components, particularly those which are stained by silver.In order to determine the cytochemical nature of the components associated with Ag-NOR proteins, the EDTA regressive preferential staining procedure for ribonucleoproteins has been applied to sections. By this means the precise localization of the Ag-NOR proteins was studied simultaneously with that of ribonucleoprotein within interphasic nucleoli and mitotic chromosomes.In interphasic nucleoli, stainable Ag-NOR proteins were localized in fibrillar centres and part of the dense fibrillar component. No silver deposits were seen on perichromatin or interchromatin fibrils and granules.In metaphasic nuclei, Ag-NOR proteins were only found on roundish fibrillar ribonucleoprotein structures, which could correspond to secondary constrictions. No silver deposits were seen on the well defined ribonucleoprotein sheet surrounding the chromosomes.In telophasic nuclei, Ag-NOR proteins were seen on the central part of roundish ribonucleoprotein fibrillar structures integrated in decondensing chromosomes. These structures have been interpreted as the nucleolar organizer regions around which rRNA synthesis resumes.In interphasic and mitotic nuclei, Ag-NOR proteins were never found within condensed chromatin but always in association with ribonucleoprotein components.The new method proposed here appears to be a useful tool for the simultaneous study of the localization of ribonucleoprotein and Ag-NOR proteins during the cell cycle.     

Microwave-stimulated glutaraldehyde and osmium tetroxide fixation of plant tissue: ultrastructural preservation in seconds

Summary Microwave-enhanced fixation of animal tissues for electron microscopy has gained in interest in recent years. Attempts to use microwave irradiation for the preparation of plant tissues are rare. In this study, I report on microwave conditions which allow a high quality preservation of plant cell structure. Tissues used were: internodes of Chara vulgaris, leaves of Hordeum vulgare, root tips of Lepidium sativum. Microwave irradiation was done with a commercial microwave oven (Sharp R-5975). Fixatives used were: 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2 and 1% osmium tetroxide in veronal/acetate buffer, pH 7.2. Conventional fixations with glutaraldehyde/osmium were compared with microwave fixations. Examinations of thin sections showed that microwave fixation (glutaraldehyde or sequential aldehyde/osmium) is an attractive and rapid alternative method for processing plant tissues for electron microscopy. The optimal conditions found were: microwave oven at power level 50 W, 6.5 ml of fixative solution, irradiation times between 32–34 s, final temperature between 40° C and 47° C.     

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.     

Ag-NOR technique in fine needle aspiration cytology of salivary gland masses.

Because of their complexity, salivary gland lesions are often difficult to identify correctly with fine needle aspiration cytology. To see whether the Ag-NOR staining technique for nucleolar organizer regions would be useful in this respect, we studied a series of smears from benign and malignant salivary gland lesions. The smears, previously treated with Papanicolaou and May-Grünwald-Giemsa stain, were destained and restained with Ag-NOR silver. The correlation between the cytologic-histologic diagnosis and the number of Ag-NORs in benign (sialadenitis, pleomorphic adenoma, oncocytoma and Warthin's tumor) and malignant lesions (adenoid cystic carcinoma, adenocarcinoma, carcinoma ex pleomorphic adenoma and squamous carcinoma) was statistically significant (P = less than .05). The Ag-NOR technique appears useful in the diagnosis of salivary gland lesions. One great advantage is that previously stained slides can be reused for silver staining, thus providing an excellent guide to the diagnosis, especially in doubtful cases and when corresponding histologic specimens or extra unstained slides are unavailable.     

Silver staining of the nucleolar organizer regions (NORs) in semithin Lowicryl sections

The one-step silver technique was applied to semithin Lowicryl sections of root meristem cells of Allium cepa and a human tumor cell line (TG cells). In vegetal cells, after 5 min of staining reaction, the Ag-NOR proteins formed ring-shaped structures peripherally within the nucleolus. In animal cells silver granules were distributed over the entire nucleolus. The specificity of the staining reaction was increased by incubation of the sections in NH4Cl and Schiff's reagent prior to Ag-NOR silver staining.     

Silver Staining of the Nucleolar Organizer Regions (NORS) in Semithin Lowicryl Sections

The one-step silver technique was applied to semithin Lowicryl sections of root meristem cells of Allium cepa and a human tumor cell line (TG cells). In vegetal cells, after 5 min of staining reaction, the Ag-NOR proteins formed ring-shaped structures peripherally within the nucleolus. In animal cells silver granules were distributed over the entire nucleolus. The specificity of the staining reaction was increased by incubation of the sections in NH₄Cl and Schiff's reagent prior to Ag-NOR silver staining.     

胃粘膜病变核仁组成区嗜银蛋白的银染计量和能谱分析

对77例胃粘膜活检标本进行银染,显示核仁组成区相关的嗜银蛋白(Ag-NOR),进行计数和统计分析,并对银染标本作电镜观察及能谱分析。结果表明,Ag-NOR 颗粒计数的多少,对胃肿瘤恶性程度的诊断有重要参考价值。电镜证实银染物质定位于核仁组成区,银染阳性区能谱分析显示 Ag 峰,说明银染反应具特异性.     

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.     

Staining Plant Cells with Silver. I. The Salt-Nylon Technique

A technique is described for selectively silver staining nucleoli, active nucleolus organizers, nucleolar material attached to chromosomes, kinetochores, synaptonemal complexes, and chromosome cores in plant cells. The technique, called salt-nylon silver staining, involves spreading cells on glass slides, treating the cells with a solution of saline sodium citrate, and incubating the cells in a silver nitrate solution covered with nylon screen. Selected variables important for achieving reliable silver staining are considered.     

Argyrophilic nucleolar organizer regions

Silver staining techniques developed to demonstrate argyrophilic nucleolar organizer regions (Ag-NORs) have been widely applied in a variety of cell kinetic studies, using the mean number of AgNORs in tumour cells as a marker for malignancy of certain types of neoplasms. However, the AgNOR techniques currently available are not entirely satisfactory, as unspecific silver precipitates readily form in the sections. On the other hand, the contrast staining, may be so weak as to render identification of the AgNORs difficult. In the present study, some of the key factors influencing the outcome of AgNOR staining were evaluated in a more systematic way. A modified AgNOR staining procedure is now proposed, giving highly contrasting AgNORs with minimal unspecific silver precipitation, thus facilitating both manual and computerized counting. The new technique involves the use of microwave irradiation in order to shorten the processing time, the use of gelatin as a protective colloid, and a Farmer's solution to optimize the specificity of the technique.