A Rapid Silver Staining and Destaining Technique for the Nucleolus Organizer Region

Silver staining of nucleolar organizing regions (NOR) is common, but a standard protocol is lacking. A modification of a rapid silver nitrate staining technique for NORs is presented here. Advantages of the modified technique include reliability, speed, cost and the fact that it can be carried out in the light.     

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 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.     

A Technique for the Simultaneous Staining of Both Nucleolar Organizer Regions and Kinetochores of Human Chromosomes with Silver

Pretreatment of human metaphase chromosomes with NaOH at a pH of 8.5, followed by staining with silver nitrate, differentially stains both the nucleolar organism regions on the 10 acrocentric chromosomes as well as the kinetochore centers on all 46 chromosomes.     

Analyses of Nucleolus Organizer Regions and Heterochromatin of Pimelodus Maculatus (Pisces, Pimelodidae)

Eighteen specimens of Pimelodus maculatus collected from Tibagi River (Sertaneja, PR, Brazil) were analyzed cytogenetically. The diploid number of 56 chromosomes was observed and karyotype was 20 M + 20 SM + 10 ST + 6 A with fundamental number (FN) of 106. Results of analyses from the nucleolus organizer regions (NORs), obtained by AgNO₃, CMA₃ and C-band staining showed marking in a terminal position on the long arm of a pair of subtelocentric chromosomes. The restriction enzyme AluI produced a linear differentiation similar to C-banding. This revised version was published online in July 2006 with corrections to the Cover Date.     

Silver Staining for Nucleolar Organizing Regions of Vertebrate Chromosomes

Refinements to a simple, one-step silver staining technique for nucleolar organizing regions are described. These include fixation of silver stained material with sodium thiosulfate and standardization of silver development conditions for different groups of vertebrates. The central advantages to the method are that it is rapid, reliable, simple, and inexpensive. Additional benefits include (i) consistent and uniform silver staining of nucleolar organizing regions, (ii) few reduced silver deposits elsewhere on the chromosomes or on the slides, (iii) generally unaltered chromosome morphology after silver treatment, and (iv) relative permanence of Permounted preparations. The method works equally well on chromosomes made from cell cultures and from solid tissues of live specimens.     

Premeiotic Change of Nucleolus Organizer Size in Neurospora

We have investigated the heritability of nucleolus organizer region (NOR) size in Neurospora crassa. By pulsed-field gel electrophoresis, we followed in genetic crosses the size of the normal or "terminal" NORs and the size of a small interstitial NOR. Tetrad analysis revealed that changes in NOR size occur frequently in the sexual phase. Moreover, most size changes occurred in the period between fertilization and meiosis, although some changes occurred during and after meiosis. Unexpectedly, increases and decreases in NOR size were not equally frequent: decreases were more common. The NOR size changes generated during meiosis were not the result of unequal crossing over between NORs on homologous chromosomes.     

Structure and variation of the Nucleolus Organizer Region in turtles

Differential staining techniques were used to study the structure and variation of the NORs of 27 species of cryptodiran turtles. No species or individuals had more than a single pair of NORs. Extensive variation in NOR structure and chromosomal location was found among higher taxa and individual variation in NOR size was common. Thirty eight percent of all individuals studied were heterozygous for the size of the NOR. However, interspecific variation in chromosomal location and structure of the NOR within major taxa was relatively rare. It is concluded that ( I ) the pattern of variation of NORs is consistent with patterns of chromosomal evolution in turtles; (2) Turtles have only a single pair of NORs whereas other animals, such as some mammals, possess numerous NORs; (3) The heterochromatin associated with the NOR is involved in the structure of the nucleolus.     

The Physical Chemistry of Silver Staining

It has been shown that silver deposition plays a part in the silver staining process. From this it has been concluded that the rate of reduction of silver within and on histological structures is an important factor.

Some factors controlling the rate of reduction, such as the adsorption of silver hydroxide and ammonia, the affinity of silver for proteins, and the protective power of the gel structures have been pointed out.

Some simple applications of the ideas to silver staining have been given and two technics described, one making use of piperidine instead of ammonia, the other carrying out the reduction in the presence of the silver solution to facilitate deposition.     

Some Introductory Comments on Silver Staining

Silver staining has become a versatile method for the visualization of specific cell structures and products. The similarity of the impregnation “nuclei” of reduced silver staining to the silver “specks” or “nuclei” of the latent image in photography is noted. “Physical” development (reduction of ionic silver in solution) in silver staining as compared to “chemical” development (reduction of ionic silver remaining in a silver halide crystal) in photographic procedures is briefly discussed.     

The Use of Protein Silver for Staining Protozoa

When commercially prepared silver products suitable for staining protozoa by the Bodian silver technic apparently became unavailable, a substitute for Protargol was prepared as follows: 0.9 g. of gelatin is dissolved by heat in 100 ml. of distilled water; to this 0.1 g. of silver nitrate is added at 60°C; this solution is poured into Columbia staining dishes (10 ml.) in which one or two drops of M/10 sodium hydroxide have been added. Copper is not used in the impregnating bath. Smears fixed in Hollande's or Schaudinn's fixatives are bleached and impregnated for 36 hours or more at 35°C. Impregnated smears are reduced with a mixture of hydroquinone and sodium sulfite, and toned with gold chloride as recommended by Kirby (1945).     

高碘酸-硝酸银染色法鉴定糖蛋白

对传统的糖蛋白染色方法(高碘酸-Schiff法)进行了改进。蛋白质的氧化采用高碘酸法,染色时采用硝酸银染色法。此方法灵敏度是高碘酸-Schiff法的100倍,而且比高碘酸-Schiff法节省16~18h。     

应用微波辐射促进组织块浸银染色

目的：探索微波辐射对组织块浸银染色各步骤的作用及微波在其中的应用方法。方法：在组织块浸银染色过程中,对组织块的固定,浸银,还原等步骤进行不同强度的微波辐射处理。结果：微波辐射明显促进了浸银,还原作用,应用适宜强度的微波辐射染色效果比常规染色有更多的优点。结论：恰当地应用微波辐射缩短组织块浸银染色时程,切实可行。     

睾酮对大鼠再生肝细胞核仁组织区影响的初步研究

以银染核仁组织区(argymphil nucleolar organiting regions,AgNORs)阳性颗粒数为指标,结合放射免疫技术,研究了大鼠部分肝切除(partial hepatectomy,PH)后,睾酮对余留肝细胞核仁组织区的影响。结果显示,pH后0～24h,各种方式(假手术、皮下注射芝麻油或睾酮)处理组,AgNORs数都下降,随后持续升高。在PH后48h和72h,低剂量(0．5ms／kg体重)睾酮对AgNORs数影响最显著,都明显高于其它两个睾酮处理组;中等剂量(2．5ms／kg体重)和高剂量睾酮(5ms／kg体重)处理对AgNORs数的影响与芝麻油处理组相比都无显著差异,但均高于假手术组;假手术后72h的AgNORs恢复到术前水平。血清睾酮含量测定显示,芝麻油处理组PH后0～48h睾酮持续下降,48h后不再下降,48h和72h时的睾酮浓度显著低于假手术组;低剂量处理,血清睾酮浓度在PH后0～24h下降,然后持续上升,在48h、72h达到假手术组的2～3倍;中等剂量和高剂量处理组,血清睾酮在PH后0～72h持续升高,是假手术组的6～7倍。以上结果初步表明,睾酮对PH后肝细胞中AgNORs的影响有两种情况：(1)在血清中的浓度是生理水平的2～3倍时,起促进作用;(2)6～7倍于生理水平时,基本无作用。     

Effect of Inflammatory Stimuli on the Silver Staining Pattern of the Rat Carotid Endothelium

Silver nitrate stains the intercellular junctions of the endothelium and other cytoplasmic or membrane components. Two protocols are described for the silver staining of rat carotid endothelium that exclude the use of pressurized fixatives and simplify the technique previously described for rat aorta. The entire surface of the carotid endothelium was examined and several parameters (stigmata, granularity, clustering of anionic sites, transversal lines, weakening of silver lines and leukocyte adhesion) were evaluated. We studied the pattern of silver staining in two situations: (1) endothelial activation and (2) neurogenic inflammation. Endothelial activation was produced by the intravenous administration of a proinflammatory albumin or polyinosinic acid. Both products cause a marked increase in leukocyte adhesion concomitant with a decrease in argyrophilia and a weakness or loss of silver lines. Neurogenic inflammation, which is mediated by substances released from sensory nerves, was induced by the intravenous administration of substance P or capsaicin. Both stimuli produced an increase in argyrophilia and weakness or loss of silver lines. Substance P caused a clustering of anionic sites, whereas this phenomenon was more discrete with capsaicin. Nearly 80% of all examined rats (controls and inflammatory stimuli treated) showed endothelial membrane disruptions formed by clusters of cells often in the shape of streaks aligned with the long axis of the vessel. The detection of these discontinuities is important, as loss of endothelial integrity is central in the initiation of pathological events.     

The Nucleolus Organizer Region of Maize (ZEA MAYS L.): Tests for Ribosomal Gene Compensation or Magnification

Ribosomal gene compensation and magnification that might be detected on a whole-plant basis was not found in maize. Plants monosomic for chromosome 6 (the NOR chromosome) were compared with monosomic-8 and monosomic-10 plants, disomic sibs, and parental lines. Assuming no rDNA compensation, monosomic-6 plants showed approximately the decrease expected in rRNA cistron number. Monosomic-8 had a normal ribosomal gene number, while monosomic-10 showed a decrease; but further documentation is needed. Besides demonstrating the absence of gene compensation, the results document our previous conclusion that maize chromosome 6 carries DNA complementary to ribosomal RNA. Further documentation was provided from studies with trisomic chromosome 6 plants showing proportional increases in ribosomal gene number. Progeny of the monosomic plants crossed as males to a standard singlecross hybrid possessed expected ribosomal gene numbers suggesting the lack of ribosomal gene magnification.—The ragged (rgd) mutant of maize, suspected of being deficient in rRNA cistrons, had a normal number.     

Synthesis of Silver Proteinates for Neurological Staining

Soluble derivatives of the AgNO₃ precipitates of various split protein products were prepared by dissolving the precipitate in a 30-40% aqueous solution of pharmaceutical peptone (Cudahy). The split proteins used included pepsin, trypsin and papain digests of albumin, globulin, gelatin, casein, protamine, and tissue proteins from heart, liver and brain; also, an Escherichia coli digest of casein, and commercial products: Amigen (Mead), casein hydrolysate (Squibb) and pharmaceutical peptone (Cudahy). Staining reactions of the silver derivatives were tested on mammalian nervous tissue. The objective of finding a silver-protein compound that stained axis cylinders selectively was attained only with redissolved silver precipitates of pharmaceutical peptone and bacterially digested casein. It was concluded that the manner of degrading a protein prior to combining it with silver was the most important factor in determining the subsequent staining reaction.