Methods for the Demonstration of Lipid Applied to Compact Bone

Sections of compact bone were cut from the diaphysis of the femur, tibia, and humerus from dogs and monkeys. These sections were either ground thin and decalcified, or decalcified and subjected to frozen sectioning. Decalcification of the sections was effected by immersion in either Decal, 10% formic acid, 10% formic acid-sodium citrate (pH 4.5) or 20% aqueous EDTA. Sections were routinely stained with oil red O, Sudan black B, or Fettrot 7B. In addition, Nile blue A and phosphine 3R were also employed. Sections stained with phosphine were viewed with a fluorescence microscope. Control sections were extracted with lipid solvents prior to application of the staining procedures. The results indicate that lipid is present in compact bone within the osteocytes, lacunae, canaliculi, and organic matrix. The significance of the lipid in these sites, particularly extracellularly, is unknown.     

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.     

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.     

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.     

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.     

Nucleolar organizer regions in meningiomas. Correlation with histopathologic malignancy grading, DNA cytometry and clinical outcome

Eighty-one meningiomas (63 grade I, 9 grade II and 9 grade III) and 2 meningeal sarcomas (grade IV) were investigated by a simple one-step silver staining for nucleolar organizer region (NOR)-associated proteins (AgNOR technique) and by DNA cytometry. The number of NORs per cell and the NOR area per cell were correlated with the histopathologic grading, as were the 5c exceeding rate and the 2c deviation index (2cDI) obtained by DNA cytometry. The differences in NOR parameters were only significant (at P less than .001) between grades I and II; P was less than .05 for the 2cDI between grades I and II. No significant differences between grades II and III were found. Among recurrent tumors, the AgNOR technique revealed the proliferative potential in 8 of 11 tumors studied, whereas DNA cytometry failed to recognize malignant features in 8 of 10 tumors investigated.     

Improvement in the specificity of the silver staining technique for AgNOR-associated acidic proteins in paraffin sections

Some recently developed silver staining methods allow selective staining of acidic nucleolar proteins. Pretreating deparaffinized sections with Schiff's reagent improves the specificity of Goodpasture and Bloom's AgNOR staining (as modified by Kodama et al.) after aldehyde fixation.     

Improvement in the Specificity of the Silver Staining Technique for Ag NOR-ASSOCIATED Acidic Proteins in Paraffin Sections

Some recently developed silver staining methods allow selective staining of acidic nucleolar proteins. Pretreating deparaffinized sections with Schiffs reagent improves the specificity of Goodpasture and Bloom's AgNOR staining (as modified by Kodama et al.) after aldehyde fixation.     

AgNOR count in resting cells (resting NOR) is a new prognostic marker in invasive bladder tumor.

PURPOSE: We have previously demonstrated that the AgNOR count in proliferating cells is a predictor of tumor recurrence in superficial bladder tumor (J. Urol. 162 (1999), 63-68). In the present study, we evaluate the type of AgNOR associated with cell cycles as a prognostic factor in invasive bladder tumor using a double staining technique employing both AgNOR and MIB-1 labelling. MATERIALS AND METHODS: Forty-four paraffin sections of invasive bladder tumors were stained simultaneously with AgNOR and MIB-1. The number of AgNORs in proliferating (MIB-1 positive) or resting (MIB-1 negative) cells were counted from a total of 100 nuclei. Correlations between MIB-1 associated AgNOR count and clinicopathological parameters were statistically analyzed. RESULTS: The AgNOR count in proliferating cells (proliferating NOR) was significantly higher than that in resting cells (resting NOR) (p<0.01). The resting NOR in tumors with distant metastases was significantly higher than that in tumors without metastases (p<0.05). Patients with a low resting NOR tumor had a better prognosis than those with a high resting NOR tumor, whereas the proliferating NOR was not associated with survival. Survival analysis revealed that the resting NOR was the most powerful prognostic marker in patients with invasive bladder tumor (p<0.05). CONCLUSIONS: Resting NOR had a predictive value in the prognosis of patients with invasive bladder tumor.     

Improved microradiographic contrast for bone stain-historadiography.

Microradiographs of 5-micron sections of methyl methacrylate embedded undemineralized bone show poor resolution, but prestaining with silver nitrate increases the radioopacity of the calcified tissues to soft x-rays without masking regional differences in microscopic mineralization. Identical results are achieved using aqueous (pH 5.8 and 7.5) or ammoniacal solutions (pH 7.9). Atomic absorption spectrometry detected no loss of calcium from the sections during staining. Osteoid in plastic-embedded bone is not rendered radiopaque by this technique even after prolonged staining times (5 min-2 hr).     

Improved Microradiographic Contrast for Bone Stain-Historadiography

Microradiographs of 5-micron sections of methyl methacrylate embedded undemineralized bone show poor resolution, but prestaining with silver nitrate increases the radioopacity of the calcified tissues to soft x-rays without masking regional differences in microscopic mineralization. Identical results are achieved using aqueous (pH 5.8 and 7.5) or ammoniacal solutions (pH 7.9). Atomic absorption spectrometry detected no loss of calcium from the sections during staining. Osteoid in plastic-embedded bone is not rendered radiopaque by this technique even after prolonged staining times (5 min-2 hr).     

Identification and characterization of sex-linked proteins of Schistosoma mansoni.

The proteins of adults worms (male and female) of two isolates (BH and RJ) of Schistosoma mansoni were extracted using Triton X-114 phase separation. The SDS-polyacrilamide gel electrophoresis profiles of the three phases (detergent, aqueous and insoluble proteins) obtained were compared after Coomassie blue and silver staining, surface radioiodination and Western blotting. No major differences were detected between the 2 isolates. Of the 25 or more proteins which partitioned into the detergent phase, only about 8 proteins could be surface radiodinated on live adult worms. A comparison was also made between the profiles of male and females worms, isolated from bisexually infected mice. Two major female-specific and one male-specific band were detected by silver and/or Coomassie staining. The female bands, 32 KDa and 18 KDa, partitioned into the detergent and aqueous phase, respectively. The male-specific band of 42 KDa remained in the insoluble phase. Antigenic differences between male and females proteins were detected by Western blotting using a sera from infected Nectomys squamipes.     

Silver staining of the nucleolar organizer regions (NORs) on Lowicryl and cryo-ultrathin sections

Nucleolar organizer region (NOR) silver staining was applied to sections of fixed material. A positive reaction on cryo-ultrathin sections was found as well as on semithin and ultrathin Lowicryl sections. Repeatable staining that was easy to control was obtained by a one-step procedure after aldehyde-Carnoy fixation. Fixation of the material by formaldehyde and glutaraldehyde alone in cacodylate buffer also maintained reaction selectivity when ammonium chloride was used after fixation. Enzymatic digestion by pronase, RNase A, DNase I, or micrococcal nuclease was applied to ultrathin Lowicryl sections. Pronase digestion removed the silver-stained proteins, whereas digestion by the nucleases did not. A routine procedure is proposed for easy NOR silver staining of sections that preserves a good tissue ultrastructure and is also compatible with cytochemical and immunological investigations.     

Staining of Nervous Tissue by Protein-Silver Mixtures

A staining method for nerves in paraffin sections is described in which an egg albumen-silver nitrate mixture is the impregnating solution. Blocks of tissue are fixed in Bouin's fixative, formol, Huber's fixative or formol-acetic-alcohol, and decalcified if necessary in Bensley's decalcifier. Sections are impregnated overnight, in the dark, at 37-56°C in a solution containing 50 ml of filtered, aqueous 0.5% dried egg albumen with 1.8-2.5 ml of 2% silver nitrate and adjusted to pH 8.2-8.3 by the addition of ammonia. The sections are then rinsed in distilled water and the silver reduced in a mixture of hydroquinone, 1 gm; anhydrous sodium sulfite, 10 gm and distilled water, 100 ml. The remainder of the process consists of washing, gold toning, fixing in 5% sodium thiosulfate, washing, dehydrating, clearing and mounting. Casein may be used as an alternative to egg albumen in the impregnating solution (0.5% casein, 50 ml; 2% silver nitrate, 1 ml). The pH value of the solution may be adjusted by a boric acid-borax buffer or ammonium hydrogen tetraborate in the place of ammonia.     

Nonrandom distribution of metaphase AgNOR staining patterns on human acrocentric chromosomes.

The metaphase nucleolar organizer regions (NORs) contain ribosomal genes associated with proteins such as upstream binding factor (UBF) and RNA polymerase I (RPI). These genes are clustered in 10 loci of the human acrocentric chromosomes (13, 14, 15, 21, and 22). Some NOR-associated proteins, termed AgNOR proteins, can be specifically stained by silver. In this study we took advantage of technical advances in digital imaging, image restoration techniques, and factorial correspondence analysis (FCA) to study the different AgNOR staining patterns of metaphase chromosomes in human lymphocytes. Three predominant patterns could be distinguished: pair (47%), stick-like (28%), and unstained (18%) structures. By studying the frequency of occurrence of each pattern on different chromosomes, two groups could be defined. Chromosomes 13, 14, and 21 carried predominantly pair or stick-like AgNOR structures, whereas chromosomes 15 and 22 mainly carried pair AgNOR structures or remained unstained. We suggest that the different AgNOR shapes reflect both the number of ribosomal genes carried by each chromosome and the differential recruitment of active ribosomal genes in each NOR cluster. This is the first study showing a nonrandom distribution of AgNOR shape among acrocentric chromosomes.     

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.     

Sensitive colloidal metal (gold or silver) staining of protein blots on nitrocellulose membranes

A sensitive staining method for protein blots on nitrocellulose membranes is described and compared with commonly used dye staining methods. It uses colloidal metal sols (gold or silver) stabilized with Tween 20 and adjusted to pH 3. It is based on the selective high-affinity binding of colloidal metal particles to the proteins and produces a red-purplish color (gold) or dark grey (silver). The sensitivity of this new staining method is in the same range as silver staining of polyacrylamide gels and matches the sensitivity of overlay assays. It will therefore be a useful tool for correlating the position of bands or spots of proteins detected with overlay assays with the complete electropherogram in a duplicate protein blot.     

Cytotaxonomy of Astyanax (Characiformes,Characidae) from the Upper Iguaçu River Basin: confirmation of the occurrence of distinct evolutionary units

Taxonomic studies of the genus Astyanax from the Iguaçu River (Brazil) indicate that they may be differentiated into 11 distinct species, some of which have not been formally described and named so for. This study focuses on three of these species, Astyanax sp. B, Astyanax sp. C and Astyanax sp. D from the Upper Iguaçu River Basin. Comparative cytogenetic analyses of C‐banding, Ag‐NORs (silver nitrate stained nucleolar organizer region) and 18S and 5S rDNA corroborate that they are distinct species. A diploid number of 50 chromosomes and similar karyotypic formulae were observed in the three taxa, with the exception of Astyanax sp. D that differs in the number of submetacentric and subtelocentric chromosomes. However, the NOR silver‐staining pattern, the heterochromatic bands (C‐bands) and the mapping of the 18S and 5S rDNA sites in the chromosomes showed divergences between all three species under study, supporting the occurrence of distinct evolutionary units.     

High-resolution image-based simulation reveals membrane strain concentration on osteocyte processes caused by tethering elements

Osteocytes are vital for regulating bone remodeling by sensing the flow-induced mechanical stimuli applied to their cell processes. In this mechanosensing mechanism, tethering elements (TEs) connecting the osteocyte process with the canalicular wall potentially amplify the strain on the osteocyte processes. The ultrastructure of the osteocyte processes and canaliculi can be visualized at a nanometer scale using high-resolution imaging via ultra-high voltage electron microscopy (UHVEM). Moreover, the irregular shapes of the osteocyte processes and the canaliculi, including the TEs in the canalicular space, should considerably influence the mechanical stimuli applied to the osteocytes. This study aims to characterize the roles of the ultrastructure of osteocyte processes and canaliculi in the mechanism of osteocyte mechanosensing. Thus, we constructed a high-resolution image-based model of an osteocyte process and a canaliculus using UHVEM tomography and investigated the distribution and magnitude of flow-induced local strain on the osteocyte process by performing fluid–structure interaction simulation. The analysis results reveal that local strain concentration in the osteocyte process was induced by a small number of TEs with high tension, which were inclined depending on the irregular shapes of osteocyte processes and canaliculi. Therefore, this study could provide meaningful insights into the effect of ultrastructure of osteocyte processes and canaliculi on the osteocyte mechanosensing mechanism.