Analysis of nucleolus organizer regions (NORs) in mitotic and polytene chromosomes ofPhaseolus coccineus by silver staining and Giemsa C-banding

Chromosomes with active nucleolus organizer regions (NORs) were visualized in root tip metaphases ofPhaseolus coccineus using the silver staining technique. A mean number of 5.5 Ag-NORs per cell was observed in 54 cells from eight plants. In the endopolyploid nuclei of the suspensor the silver technique did not demonstrate the reported specificity for nucleolus organizer activity, because there was usually pale staining of nucleoli and preferential staining of heterochromatic regions in the polytene chromosomes including pericentromeric material, telomeres and NORs. The mean number of NORs per nucleolus as detected by this method was 5.8 (28 nucleoli analysed). Using a modified preparation technique, giant chromosomes stained pale, but nucleoli of suspensor cells displayed darkly silver staining internal domains, each of which originating from a nucleolus organizer.—Giemsa C-banding of endopolyploid suspensor nuclei revealed C-positive nucleolus organizers with darkly staining intranucleolar fibrils. The latter were frequently involved in inter-NOR associations. In 34 nucleoli analysed, the mean number of Giemsa C-positive NORs per nucleolus was 6.0.Dedicated to Professor Dr.Lothar Geitler on the occasion of his 80th birthday.     

Silver staining,immunofluorescence, and immunoelectron microscopic localization of nucleolar phosphoproteins B23 and C23

Nucleolar organizer region (NOR)-specific silver staining and immunolocalization of nucleolar phosphoproteins B23 and C23 were compared in Novikoff hepatoma ascites cells. Silver staining and protein C23 immunostaining were both localized in the fibrillar shell surrounding the fibrillar center and in the fibrillar center. During mitosis, silver staining and protein C23 were localized at the NORs. Therefore, protein C23 and the silver-staining protein both seem to be associated with rDNA-containing structures (Mirre and Stahl 1981). A comparison of toluidine blue staining specific for RNA and B23 immunostaining demonstrated that protein B23 was associated with RNA-containing regions of the nucleolus and was absent from the fibrillar centers. Localization of these proteins and their functions are discussed in relation to the organization of the nucleolus.     

The application of the nucleolar organizer region silver staining (AgNOR) to backscattered electron imaging (BEI)

Until recently scanning electron microscopes were mainly used to observe surfaces. However, it has been proved that a backscattered electron detector can give an image (BEI) of the specimen's internal structure after heavy metal staining. In this paper, we report how we have applied the silver staining for NOR-associated proteins to scanning electron microscopy, studying C3H10T1/2 cells in culture. This technique allows to localize, inside the nucleus, the nucleolar arrangement of AgNOR-associated proteins. In BEI imaging, the silver staining shows several intranucleolar silver spot-like deposits sometimes associated in “doublets” as on metaphasic chromosomes. These silver grains probably represent the fibrillar centre location, thought to be the interphasic counterpart of the NORs. However, these silver spot granules are more numerous during interphase.     

The application of the nucleolar organizer region silver staining (AgNOR) to backscattered electron imaging (BEI)

Until recently scanning electron microscopes were mainly used to observe surfaces. However, it has been proved that a backscattered electron detector can give an image (BEI) of the specimen's internal structure after heavy metal staining. In this paper, we report how we have applied the silver staining for NOR-associated proteins to scanning electron microscopy, studying C3H10T1/2 cells in culture. This technique allows to localize, inside the nucleus, the nucleolar arrangement of AgNOR-associated proteins. In BEI imaging, the silver staining shows several intranucleolar silver spot-like deposits sometimes associated in "doublets" as on metaphasic chromosomes. These silver grains probably represent the fibrillar centre location, thought to be the interphasic counterpart of the NORs. However, these silver spot granules are more numerous during interphase.     

Localisation of nucleolus-organising regions in interphase cells

Summary A technique based on the use of silver solutions, which selectively stains the nucleolus-organising regions (NORs) in chromosomes, was applied to interphase Ehrlich tumour cells. The results indicate that nucleolar fibrillar centres correspond to the NORs.     

Patterns of silver staining in cells of six-day blastocyst and kidney fibroblast of the domestic rabbit

The distribution pattern of silver-NORs was studied in cells of six-day blastocysts and kidney fibroblasts of the rabbit using the Ag-AS technique. At metaphase and interphase there was a binomial distribution of the number of stained sites in both populations but blastocysts had a greater percentage of cells with larger numbers of stained sites. Up to 7 of the 8 chromosomes known to bear NORs were stained in cells from blastocysts while a maximum of 6 were stained in fibroblasts. A significant difference was found between the mean numbers of chromosomal NORs per cell in metaphases from blastocysts and fibroblasts, where they were 4.2 and 3.3 respectively. Similarly, the mean number of NORs in interphase was significantly greater in cells from blastocysts. The distribution of staining on chromosome pair 13 was related to cell type. Significantly more cells in blastocysts than fibroblasts showed staining in this chromosome pair.     

Ultrastructural localization of Ag-NOR stained proteins in the nucleolus during the cell cycle and in other nucleolar structures

EM investigation of Ag-AS-NOR staining after short glutaraldehyde prefixation followed by Carnoy fixation maintained good ultrastructural preservation and reactive selectivity. This enables exact localization of silver deposits both in the fibrillar centers of typical or segregated nucleoli during interphase, and in chromosome NORs during mitosis. These results argue in favour of the possibility that fibrillar centers are the interphasic counterpart of chromosome NORs. Special structures such as nucleolar blobs and remnants usually considered to be of nucleolar origin, were also stained. — These findings seem to indicate a relationship between the distribution of the silver-stained proteins, the arrangement of the nucleolar structures and the degree of nucleolar activity resulting from the experimental conditions. These results are of interest at the time when the concept of the nucleolar matrix is gradually emerging.     

Simultaneous immunoelectron microscopic visualization of protein B23 and C23 distribution in the HeLa cell nucleolus

The intranucleolar distribution of phosphoproteins B23 and C23 was visualized simultaneously by post-embedding immunoelectron microscopy in HeLa cell nucleoli, using specific antibodies. The data show that proteins B23 and C23 co-localize to the same nucleolar compartments, i.e., the dense fibrillar component and the granular component. Neither of the two antibodies is significantly associated with the fibrillar centers in these cells, although the fibrillar centers appear positive after silver staining. These findings suggest that other unidentified components must be responsible for the silver staining observed in the fibrillar centers of interphase nucleoli. The results are discussed in the light of previously reported data obtained by preembedding immunolabeling techniques and by silver staining, which both suggested a localization of protein C23 inside the fibrillar centers.     

Distribution of fibrillar centers and silver-stained components in the nucleolus of human Sertoli cells

The nucleolus of the human Sertoli cell consistently showed three distinct, spontaneously segregated parts: 1. one or two large, silver-positive fibrillar centers; 2. strands of dense fibrillar component continuous with the dense cords surrounding the fibrillar center. These components were also silver-positive; 3. a granular, silver-negative mass. These observations show that in the human Sertoli cell the number of fibrillar centers is far lower than the diploid number of NORs. They also suggest that the fibrillar center might contain several NORs in this cell type.     

Structural organization of chromatin in nucleolar organizer regions of nucleoli with a nucleolonema-like and compact ribonucleoprotein distribution

We have studied the relationship between the structural organization of intranucleolar chromatin and fibrillar nucleolar structures, fibrillar centers, and RNP fibrillar component, which are the interphase counterpart of metaphase nucleolar organizer regions (NORs), in regenerating rat hepatocytes and in a human tumor cell line (TG cells). These two cell types were characterized by a nucleolonema-like and compact nucleolar RNP distribution, respectively. We found that, in sections selectively stained for DNA, the intranucleolar chromatin composed of extended, nonnucleosomal DNA filaments formed roundish agglomerates with a spatial distribution which was superimposable on that of the fibrillar centers and the RNP fibrillar component around them and on sites of the silver reaction in samples selectively stained for Ag-NOR proteins. The agglomerates of extended nonnucleosomal DNA filaments were small and numerous in regenerating hepatocyte nucleoli, in which the RNP components had a nucleolonema-like distribution, whereas they were large and few in TG cell nucleoli, in which the RNP components showed a compact organization. Since the pattern of ribosomal RNA synthesis and processing was similar in the two cell types, a model was proposed in which the difference in size and shape of the agglomerates of extended DNA might be responsible for the different structural organization of the RNP components.     

Three-dimensional analysis of fibrillar centers and associated chromatin in the nucleolus of human oocytes in primordial follicles

The intranucleolar localization of fibrillar centers and their relationships with nucleolus-associated chromatin were determined in stereopairs of human oocyte nucleoli obtained by computer reconstruction of serial sections. This study showed that there was no numerical relationship between the number of fibrillar centers and the number of chromosomal NORs. The three-dimensional reconstruction demonstrated that the majority of fibrillar centers was directly connected with the nucleolus-associated chromatin.     

The three-dimensional ultrastructure of interphasic and metaphasic nucleolar argyrophilic components studied with high-voltage electron microscopy in thick sections

The three-dimensional structure of the nucleolar argyrophilic components was studied by recording stereo-pairs of tilted thick sections--0.5-2 microns thick--observed with 200 and 300 kV high-voltage electron microscopy (HVEM). Using a very specific silver staining method, the argyrophilic components were stained with a high contrast relatively to the unstained background, thus allowing their study with a high resolution within thick sections. This study was performed on compact nucleoli (of HL60 and K562 cells), on reticulated nucleoli (of human breast cancerous cells) and on metaphasic nucleolar organizer regions (NORs). In compact nucleoli argyrophilic components show a 'knotted rope-like' structure in which knots are constituted of one central fibrillar centre surrounded at some distance by loops of the dense fibrillar component and in which the rope is constituted of dense fibrillar component. In reticulated nucleoli silver deposits are confined to the surface of the nucleolonema as several strands twisted at the periphery of the fibrillar component. During metaphase some NORs get a characteristic crescent-shaped structure disposed at the periphery of some chromosomes.     

ULTRASTRUCTURAL LOCALIZATION OF ARGYROPHILIC PROTEINS IN NUCLEOLI OF ZEA MAYS BY TWO SILVER STAINING TECHNIQUES

Ag staining was applied on interphasic nucleoli of Zea mays root cells 120h after germination. We applied the two-step Ag-NOR staining technique to small root fragments and the one-step technique to sections of Lowicryl-embedded tissue. The small-sized silver grains were mainly located in the dense fibrillar component (DFC). The unstained fibrillar centers (FCs) differed in their proteinic contents from the NOR (which is positively silver stained) and were not the interphasic NOR counterpart.     

A new version of the Ag?NOR technique. A combination with DAPI staining

Summary The Ag−NOR staining technique is widely used for visualizing nucleolar organizer regions (NORs) in various plant and animal tissues. We describe a simple and time-saving combination of Ag−NOR staining with DNA detection by fluorescence microscopy. This modification was tested on cultured cells and semi-thin sections of plastic-embedded tissues. Of the different fixatives and embedding media used in our studies, the best results (i.e., high selectivity of staining, and lack of or very low background precipitation) were obtained with fixation in methanol-acetone at −20°C for cultured cells, and fixation in 4% formaldehyde followed by embedding in Histocryl resin for tissue sections. The optimal time of Ag−NOR staining was determind experimentally for all materials tested. The specificity of the staining was checked at the electron microscopical level. Especially good results were obtained by mixing epifluorescence with standard bright-field illumination. In such a combination, Ag−NOR-positive nucleoli, or their fibrillar centres and dense fibrillar components, were clearly visible against a bright background of nuclear DNA.