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.     

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.     

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.     

A Technique for Simultaneous Antikinetochore Immunofluorescence Staining and Q-Banding in Chromosomes from Human Lymphocytes

Antikinetochore immunofluorescence staining has been used in several studies to determine whether a second kinetochore is present, active, or both, in multicentric chromosomes. All of these studies have used tissue culture cells, and contended with the problem of obtaining well spread, banded metaphase chromosomes without affecting the kinetochore staining. We have adapted hypotonic, centrifugation and chromosome staining techniqnes to obtain simultaneous Q-banding and bright kinetochore staining of chromosomes from human lymphocytes.     

The Effect of Ph on Silver Staining of Nucleolus Organizer Regions

The effect of pH on silver staining of the nucleolus organizer regions (NORs) of human chromosomes has been investigated between pH 6.5 and 12.0. Nonvolatile mixtures of ethanolamine and ethanolammonium nitrate replaced the ammonia of standard procedures. The optimal NOR staining obtained at pH 3.5 by the silver staining procedure of Howell and Black served as a standard; this procedure stained all ten NORs in 90% of mitoses. Similar NOR staining was found in 75% of mitoses stained at pH 11.7 or 11.8, but only in 10-15% of mitoses stained between pH 11.6 and 10.0. Between pH 10.0 and 9.0 NOR staining was incomplete, and between pH 8.5 and 6.5 there was no NOR staining.     

A Differential Staining Technique for Simultaneous Visualization of Mitotic Spindle and Chromosomes in Mammalian Cells

A new technique has been devised for staining the mitotic spindle in mammalian cells while preserving spindle structure and chromosome number. The cells are trypsinized and fixed with a 3:1 methanobacetic acid solution containing 4 mM MgCl₂ and 1.5 mM CaCl₂ at room temperature. The cells are then placed on slides and treated with 5% perchloric acid before staining with a 10% acetic acid solution containing safranin O and brilliant blue R. The preserved spindles appear dark blue against a light cytoplasmic background with chromosomes stained bright red. Individual chromosomes and chromatids are clearly visible. Positioning of the chromosomes relative to the spindle apparatus is readily ascertained allowing easy study of mitotic spindle and chromosome behavior.     

The Three-dimensional Study of Chromosomes and Upstream Binding Factor-immunolabeled Nucleolar Organizer Regions Demonstrates Their Nonrandom Spatial Arrangement during Mitosis

The volumic rearrangement of both chromosomes and immunolabeled upstream binding factor in entire well-preserved mitotic cells was studied by confocal microscopy. By using high-quality three-dimensional visualization and tomography, it was possible to investigate interactively the volumic organization of chromosome sets and to focus on their internal characteristics. More particularly, this study demonstrates the nonrandom positioning of metaphase chromosomes bearing nucleolar organizer regions as revealed by their positive upstream binding factor immunolabeling. During the complex morphogenesis of the progeny nuclei from anaphase to late telophase, the equal partitioning of the nucleolar organizer regions is demonstrated by quantification, and their typical nonrandom central positioning within the chromosome sets is revealed.     

Electron Tomography of Metaphase Nucleolar Organizer Regions: Evidence for a Twisted-Loop Organization

Metaphase nucleolar organizer regions (NORs), one of four types of chromosome bands, are located on human acrocentric chromosomes. They contain r-chromatin, i.e., ribosomal genes complexed with proteins such as upstream binding factor and RNA polymerase I, which are argyrophilic NOR proteins. Immunocytochemical and cytochemical labelings of these proteins were used to reveal r-chromatin in situ and to investigate its spatial organization within NORs by confocal microscopy and by electron tomography. For each labeling, confocal microscopy revealed small and large double-spotted NORs and crescent-shaped NORs. Their internal three-dimensional (3D) organization was studied by using electron tomography on specifically silver-stained NORs. The 3D reconstructions allow us to conclude that the argyrophilic NOR proteins are grouped as a fiber of 60–80 nm in diameter that constitutes either one part of a turn or two or three turns of a helix within small and large double-spotted NORs, respectively. Within crescent-shaped NORs, virtual slices reveal that the fiber constitutes several longitudinally twisted loops, grouped as two helical 250- to 300-nm coils, each centered on a nonargyrophilic axis of condensed chromatin. We propose a model of the 3D organization of r-chromatin within elongated NORs, in which loops are twisted and bent to constitute one basic chromatid coil.     

癌症患者淋巴细胞核仁形成区活性变异的研究

应用银染-G带复合显示方法研究了肺癌、胃癌、肠癌、乳腺癌患者的外周淋巴细胞核仁形成区(NOR)活性。与正常对照相比,肺癌第15号染色体的Ag-NOR频率及Ag-NOR总频率增加,乳腺癌第14号染色体的Ag-NOR频率减少,胃癌第14号染色体Ag-NOR频率减少而第22号染色体的Ag-NOR频率增加。肠癌未见明显变异。结果提示不同部位的肿瘤具有不同的优势银染型,rRNA基因的表达可能存在肿瘤部位的特异性。     

Gold Toning Improves the Visualization of Nucleolar Organizer Regions in Paraffin Embedded Tissues

A modification of the silver colloid technique for staining nucleolar organizer regions in paraffin embedded tissues is described. This modification involves the application of a gold toning step with subsequent gold reduction, if necessary, following incubation of sections in the standard silver colloid solution. Silver stained nucleolar organizer regions (AgNORs) in toned sections are more sharply delineated when compared to untoned controls. in high grade tumors the addition of the toning step results in significantly higher AgNOR counts due to the ability to discriminate more easily individual AgNORs in argyrophilic aggregates within the nucleus. It is recommended, because of enhanced visualization, that this modification of the silver colloid technique be used in studies involving quantification of AgNORs in tissue sections.     

Gold Toning Improves the Visualization of Nucleolar Organizer Regions in Paraffin Embedded Tissues

A modification of the silver colloid technique for staining nucleolar organizer regions in paraffin embedded tissues is described. This modification involves the application of a gold toning step with subsequent gold reduction, if necessary, following incubation of sections in the standard silver colloid solution. Silver stained nucleolar organizer regions (AgNORs) in toned sections are more sharply delineated when compared to untoned controls. in high grade tumors the addition of the toning step results in significantly higher AgNOR counts due to the ability to discriminate more easily individual AgNORs in argyrophilic aggregates within the nucleus. It is recommended, because of enhanced visualization, that this modification of the silver colloid technique be used in studies involving quantification of AgNORs in tissue sections.     

Study of the Nucleolar Organizer Regions in Palinurus elephas (Crustacea: Decapoda)

Using fluorescence in situ hybridization (FISH), chromomycin (CMA₃) staining and silver staining, we studied the nucleolar organizer regions in the spiny lobster Palinurus elephas in order to extend our knowledge on the karyology of this commercially important species. Multiple NORs have been detected by FISH, and CMA₃ showed a good correspondence between the localization of GC-rich heterochromatin and the ribosomal genes mapped by FISH. In contrast, the number of Ag-positive regions was higher than the number of FISH and CMA₃ signals, which may be explained by silver staining of the kinetochores. A variability in the number of FISH and CMA₃ signals has been detected in metaphases I and II which is probably due to the occurrence of rDNA cistrons on B chromosomes.     

A Technic for Differential Staining of Nucleoli and Chromosomes

A procedure is described which enables a stain to be definitely located in the substance of the nucleolus. Material is fixed in either Navashin or Levitsky; the chromatin is stained by means of the improved Feulgen technic introduced by de Tomasi, and preparations brought thru the washing solutions down to distilled water. From distilled water the material is transferred to a mordant solution, 5% sodium carbonate in water, in which it is left for at least one hour. After mordanting wash well with water then stain for ten minutes in light green solution (90% alcohol, 100 cc, light green SFY, 0.5 g, aniline oil, 2 drops, well shaken); differentiate in alcoholic sodium carbonate solution, (70% alcohol saturated with carbonate); treat with 95% alcohol, absolute alcohol, equal parts xylene and absolute alcohol, clear in pure dry xylene and mount in neutral balsam. Cytoplasm and karyolymph should be quite clear, with magenta chromatin and well defined green nucleoli. The light green does not behave like a simple counterstain as in previous technics but as a definite stain for nucleolar material.     

Staining of Ultraviolet Irradiated Chromosomes by the Gomori Alkaline Phosphatase Technique

When a chromosome segment is selectively irradiated with an ultraviolet micro-beam, the chromosome(s), which normally appear black by medium-dark phase-contrast microscopy, become “pale” in the irradiated region (decrease in refractive index). Previous ultraviolet absorption and Feulgen staining studies indicated that all or most of the deoxyribonucleic acid is lost in this region. After fixation, the irradiated area appears pale with most of the usual staining methods. The residual material in the paled spot, however, can be stained with the Comori alkaline phosphatase technique and is seen to be directly continuous with the nonirradiated segments. With the bright field microscope, there appears to be no decrease or increase in chromosome width. It is concluded that staining by the Gomori technique is independent of the presence or absence of deoxyribonucleic acid. Positive staining of chromosomes by the nonenzymatic peroxide method of Danielli indicated that staining was due to nonspecific precipitation of calcium phosphate rather than to enzymatic activity.     

微卫星DNA聚丙烯酰胺凝胶电泳(PAGE)银染法的改良

为了筛选和检测绵羊种群中具有较好表现的微卫星多态性,采用Touch-down PCR方法扩增绵羊基因组上的微卫星DNA序列后,经聚丙烯酰胺凝胶电泳分离,采用改良的银染法对微卫星位点进行多态性检测,染色结果与常规银染法相比,该方法具有灵敏度高、背景浅、污染小、条带清晰等突出特点,能有效地控制染色背景,显著提高检测分辨率,整个染色过程所需时间短,具有广泛的推广价值。     

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.     

A Mordanting Fixation for Intense Staining of Small Chromosomes

A combination iron-mordant fixative in which propionic acid is substituted for acetic acid has been found useful in preparing small plant chromosomes for carmine stained squashes. Propionic acid is better than acetic acid because it holds more iron in stable solution. The fixative is a 3:1 mixture of 95% alcohol and pure propionic acid which contains 400 mg. of Fe(OH)₃ per 100 ml. of propionic acid. The latter is previously prepared by dissolving the dry freshly prepared Fe(OH)₃ in it. To each 10 ml. vial of fixative is added a few drops of carmine stain. Standard aceto-carmine squashes of material fixed in this mixture show quick intense staining and are especially useful for differentiated chromosomes at mitotic prophase.