Selective staining of the same set of nucleolar phosphoproteins by silver and Giemsa

Gel electrophoresis of nucleolar isolates from Zajdela ascites hepatoma cells followed by various staining procedures revealed a common set of bands that stained selectively with silver and Giemsa. The gel bands, corresponding to molecular weights of 104, 78, 37, and 29 kilodaltons (kd), appeared to contain phosphoproteins that were at least partly associated with oligo-deoxyribonucleotides. Enzyme digestion studies showed that the Giemsastainability was due to the phosphorylated state of the proteins. The positive selective silver-staining reaction in gels could be most likely attributed to the high content of carboxyl groups present in these phosphoproteins. The significance of these findings in relation to cytological results produced by selective silver staining of nucleolus organizing regions (NORs) and by Giemsa N-banding is discussed.     

Modified silver staining of nucleolar organizer regions to improve the accuracy of image analysis

Silver staining of nucleolar organizer regions (NORs) and their subsequent quantification by image analysis are used increasingly in human pathological specimens and experimental models. Because certain conditions determined by the type of tissue and/or its fixation render AgNOR segmentation for image analysis difficult due to insufficient contrast or nonspecific silver precipitation, we propose three improvements to the original technique to overcome these difficulties. Pretreatment with 7% nitric acid produced very distinct dark brown images of AgNORs on a yellow background. The gradient of background colors allowed easy discrimination of nucleolar, nuclear and cytoplasmic structures. Seven morphometric parameters related to number, size and shape of AgNORs were evaluated quantitatively by image analysis on sections pretreated with nitric acid and on adjacent sections treated with citrate buffer in a wet autoclave according to the most widely accepted method for image analysis of AgNOR. Both methods yielded similar results. A second improvement was achieved by coating the slides with 7% celloidin solution in ethyl alcohol-ether prior to AgNOR staining and acid pretreatment. This coating prevented nonspecific silver deposition on argyrophilic bacteria and other tissue debris in human vaginal smears that could make visualizing AgNOR sites difficult. Finally, placing sections face down on the staining solution prevents the formation of nonspecific silver precipitates. These procedures can be applied together or separately according to the requirements of the material to be evaluated.     

Modified silver staining of nucleolar organizer regions to improve the accuracy of image analysis

Silver staining of nucleolar organizer regions (NORs) and their subsequent quantification by image analysis are used increasingly in human pathological specimens and experimental models. Because certain conditions determined by the type of tissue and/or its fixation render AgNOR segmentation for image analysis difficult due to insufficient contrast or nonspecific silver precipitation, we propose three improvements to the original technique to overcome these difficulties. Pretreatment with 7% nitric acid produced very distinct dark brown images of AgNORs on a yellow background. The gradient of background colors allowed easy discrimination of nucleolar, nuclear and cytoplasmic structures. Seven morphometric parameters related to number, size and shape of AgNORs were evaluated quantitatively by image analysis on sections pretreated with nitric acid and on adjacent sections treated with citrate buffer in a wet autoclave according to the most widely accepted method for image analysis of AgNOR. Both methods yielded similar results. A second improvement was achieved by coating the slides with 7% celloidin solution in ethyl alcohol-ether prior to AgNOR staining and acid pretreatment. This coating prevented nonspecific silver deposition on argyrophilic bacteria and other tissue debris in human vaginal smears that could make visualizing AgNOR sites difficult. Finally, placing sections face down on the staining solution prevents the formation of nonspecific silver precipitates. These procedures can be applied together or separately according to the requirements of the material to be evaluated.     

Visualization of nucleolar organizer regions in mammalian chromosomes using silver staining

A simple ammoniacal silver staining procedure, designated Ag-AS, differentially stains the chromosomal locations of ribosomal DNA in certain mammalian species. This was critically demonstrated by Ag-AS staining of the nucleolus organizer regions in karyotypes of the same species and cell lines used for locating the ribosomal cistrons by DNA/RNA in situ hybridization. With Ag-AS, silver stained NORs (Ag-NORs) are visualized as black spherical bodies on yellow-brown chromosome arms. Ag-NORs were visualized throughout mitosis at the secondary constrictions in the rat kangaroo, Seba's fruit bat, Indian muntjac, and Rhesus monkey. The Chinese hamster and cattle have telomeric Ag-NORs, the mouse subcentromeric Ag-NORs, and the field vole Ag-NORs as minute short arms or choromosomal satellites. Ag-NORs occur at both secondary constrictions and at telomeres in the cotton rat. Variability in Ag-NOR pattern included differences in the number of Ag-NORs per cell within a cell population, size of Ag-NORs among chromosomes of a complement, and presence of Ag-NOR on particular chromosomes in two cell lines of the Chinese hamster. The available cytochemical data suggest that the Ag-AS reaction stains chromosomal proteins at the NOR rather than the rDNA itself.     

Preferential association of nucleolar organizing human chromosomes as revealed by silver staining technique at mitosis

Summary The frequency of different types of satellite associations of nucleolar organizing human chromosomes (i.e. acrocentric chromosomes; 13, 14, 15, 21, and 22) is reported using 10 normal individuals by Ag-staining technique. The preferential involvement of acrocentric chromosomes in satellite association is suggested. Only acrocentric chromosomes with active NORs (i.e. Ag-stained) were found in association while unstained (inactive NORs) chromosomes were never seen in satellite association. In general as number of NORs expression increase, the frequency of association per cell was also increased. A possible mechanism and the clinical consequences of such an unusual phenophenon is described.     

Proteins C23 and B23 are the major nucleolar silver staining proteins.

To examine the silver staining proteins of Novikoff hepatoma nucleoli, the nucleolar proteins were separated on two-dimensional polyacrylamide gels with an isoelectric focusing first dimension and an acid-urea gel second dimension. The nucleoli were sequentially extracted with (1) 0.6 M potassium acetate, pH 5.5 and (2) 2 M potassium acetate — 5 M urea — 10 mM Tris, pH 7.5. The silver staining method used for the detection of silver binding proteins in gels was similar to that used to stain the nucleolar granules on microscope slides. Two major silver staining proteins were found which were identified as (molecular weight × 10^?3/pI) proteins C23 (100/5.3) and B23 (37/5.1). These two proteins are the major acidic proteins in Novikoff hepatoma nucleoli.     

Identification of a silver binding protein associated with the cytological silver staining of actively transcribing nucleolar regions.

Nucleoli isolated from Novikoff hepatoma cells were stained with AgNO3 to demonstrate the typical staining of active ribosomal cistrons. Pre-treatment of the nucleoli with 80 mM Tris-HCl (pH 7.5) -- 2.0 M NaCl did not interfere with silver staining. Treatment of the nucleoli with 80 mM Tris-HCl (pH 7.5) -- 0.15 M NaCl did, however, eliminate silver binding. Serial extraction of nucleoli with 2.0 M NaCl buffer followed by 0.15 M NaCl buffer also abolished silver staining. Analysis of the supernatant fraction of these extracts by polyacrylamide gel electrophoresis indicates that, although more than one nucleolar protein can bind silver, only one protein is associated with the staining of active ribosomal cistrons.     

Microwave irradiation improvements in the silver staining of the nucleolar organizer (Ag-NOR) technique

The well-know technique of silver staining of the nucleolar organizer (Ag-NOR) is improved in contrast, selectivity and speed when performed with microwave irradiation. The Ag-NOR technique is a very useful tool for studies on the functional morphology and molecular architecture of the nucleolus, and is reputed to be one of the best techniques for diagnosis and prognosis of cancer lesions. To test the generality of the enhancing effects. our study has involved the use of both mammalian and plant cells. Two steps in the process are improved quantitatively by microwave irradiation: fixation and staining itself. Fixation with the ethanol-based reagent, Kryofix, for 3 min in the microwave oven, resulted in good structural preservation at the optical level, and enhanced the contrast and selectivity of silver staining. On the contrary, we found that neither glutaraldehyde fixation, nor a treatment of sections with Carnoy's solution, improved Ag-NOR staining. After an analysis of the effects of the different substances involved in sample preparation, we conclude that ethanol is an essential factor for fixation for nucleolar staining, particularly if aldehydes are eliminated from fixative solutions. The process of staining was performed with a drop of staining solution on a semithin section of plastic-embedded tissue intthe microwave oven for 1 min. Staining under these conditions always improved the visualization of nucleoli, regardless of the fixation procedure. Therefore, microwave irradiation at both steps is recommended for giving the best results. Microwave irradiation probably enhances fixation by controlled heat, whereas the increase in reactivity of the staining solution is a direct effect by the microwaves on the silver ions themselves. We used this method to study nucleolar materials during mitosis in proliferating plant cells. Current applications of Ag-NOR staining can be improved with this technical modification.     

An improved technique for selective silver staining of nucleolar organizer regions in human chromosomes

Summary A reliable technique for staining human chromosomal nucleolar organizers (NOR's) with silver solutions is described. The NOR's can be selectively stained dark brown by silver solutions leaving the chromosome arms unstained and available for counterstaining with orcein or Giemsa dyes. Unequivocal identification of chromosome pairs bearing NOR's can be achieved using fluorescent banding techniques followed by silver staining. The silver staining procedure for NOR's was simplified and standardized through control of the chemical and physical conditions during silver impregnation and developing.     

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.     

用银染mRNA差异显示进行基因筛选

介绍一种简便、快速的mRNA差异显示法银染法。并对在逆转录时总RNA加入的量、dNTP的浓度、银染的条件、差异条带的回收及再扩增等方面作了探讨和改进。实验表明,用银染进行mRNA差异显示具有快速、简便、经济、可靠性高等优点。为新基因的发现和克隆提供了有用的手段。