Staining of intracellular deposits of uranium in cultured murine macrophages

In our studies of the health effects of internalized depleted uranium, we developed a simple and rapid light microscopic method to stain specifically intracellular uranium deposits. Using J774 cells, a mouse macrophage line, treated with uranyl nitrate and the pyridylazo dye 2-(5-bromo-2- pyridylazo)-5-diethylaminophenol, uranium uptake by the cells was followed. Specificity of the stain for uranium was accomplished by using masking agents to prevent the interaction of the stain with other metals. Prestaining wash consisting of a mixture of sodium citrate and ethylenediaminetetraacetic acid eliminated staining of metals other than uranium. The staining solution consisted of the pyridylazo dye in borate buffer along with a quaternary ammonium salt, ethylhexadecyldimethylammonium bromide, and the aforementioned sodium citrate/ethylene-diaminetetraacetic acid mixture. The buffer was essential for maintaining the pH within the optimum range of 8 to 12, and the quaternary ammonium salt prevented precipitation of the dye. Staining was conducted at room temperature and was complete in 30 min. Staining intensity correlated with both uranyl nitrate concentration and incubation time. Our method provides a simple procedure for detecting intracellular uranium deposits in macrophages.     

Staining of intracellular deposits of uranium in cultured murine macrophages

In our studies of the health effects of internalized depleted uranium, we developed a simple and rapid light microscopic method to stain specifically intracellular uranium deposits. Using J774 cells, a mouse macrophage line, treated with uranyl nitrate and the pyridylazo dye 2-(5-bromo-2- pyridylazo)-5-diethylaminophenol, uranium uptake by the cells was followed. Specificity of the stain for uranium was accomplished by using masking agents to prevent the interaction of the stain with other metals. Prestaining wash consisting of a mixture of sodium citrate and ethylenediaminetetraacetic acid eliminated staining of metals other than uranium. The staining solution consisted of the pyridylazo dye in borate buffer along with a quaternary ammonium salt, ethylhexadecyldimethylammonium bromide, and the aforementioned sodium citrate/ethylene-diaminetetraacetic acid mixture. The buffer was essential for maintaining the pH within the optimum range of 8 to 12, and the quaternary ammonium salt prevented precipitation of the dye. Staining was conducted at room temperature and was complete in 30 min. Staining intensity correlated with both uranyl nitrate concentration and incubation time. Our method provides a simple procedure for detecting intracellular uranium deposits in macrophages.     

Pyrogallol red-vanadium complex—a new stain for electron microscopy

 We report on the application of a pyrogallol red-vanadium complex (PR-V) for ultracytochemical staining of proteinaceous structures in animal tissues and cell cultures. This dye may be used as a general purpose stain in electron microscopy. In contrast to osmium tetroxide, the price of the material is low and no toxic vapors are produced. The PR-V complex was prepared by addition of vanadium (IV) oxide sulfate to pyrogallol red dissolved in acetate buffer (pH 5.6). The formation of the complex was indicated by a color change from purple-red (λ_max=520 nm) to violet (λ_max=539 nm) which occurred at equimolar concentrations of the dye and the metal salt. Under these conditions PR-V was stable for several days. The mechanism of PR-V binding was checked in dot blots using different proteins as well as heparin for control. While heparin remained unstained, proteins were stained in a dose-dependent manner. Deamination of proteins with nitric oxide strongly reduced PR-V staining in dot blots as well as in cell cultures. Optimal staining results of animal cells and tissues were obtained in specimens that had been mildly fixed for at least 1 h or longer with a mixture of 0.1% glutaraldehyde and 1.0% paraformaldehyde dissolved in phosphate-buffered saline, pH 7.2, washed with acetate buffer, pH 5.6, and subsequently treated with PR-V in the presence of 50% ethanol at room temperature. Control specimens without PR-V but treated en bloc with uranyl acetate or sodium molybdate showed similar contrast but less details in the ultrastructure of the tissue. All specimens were embedded in epoxy resin and ultrathin sections were stained conventionally with uranyl and lead salt solutions. In electron micrographs, membrane-associated particles, stress fibers and filaments of the cell cortex, collagen fibrils, tight junctions and desmosomes, and other proteinaceous components were clearly visualized only in the PR-V-treated specimens. In conclusion, the ability to bind selectively and specifically to proteinaceous structures makes PR-V a versatile stain to study the localization and distribution of these structures in cells and tissues at the ultrastructural level. Accepted: 14 June 1996     

Bismuth Hematoxylin Stain for Arginine Residues

Bismuth ions complex with hematoxylin oxidized by sodium iodate to form a dark blue dye that stains structures with high arginine content. In citrate buffer at pH 5.2, staining is confined to cell nuclei and myelin sheaths. Extraction of nucleic acids has little effect on the stain. Blockade of the guanidino groups of arginine completely abolishes staining.     

Sulfated acid mucopolysaccharides in the cortical granules of eggs. Effects of quaternary ammonium salts on fertilization

Sulfated acid mucopolysaccharides have been shown to be constituents of cortical granules in sea urchin and vertebrate eggs. These observations were made possible by retaining soluble acid mucopolysaccharides in situ within the eggs by precipitation during fixation with cetyltrimethyl-ammonium bromide, a quaternary ammonium salt. The sulfated mucopolysaccharides were then identified by staining with Astrablau at pH 0.2 and also by reaction with sodium rhodizonate. Staining reaction with Alcian blue at pH 2.5 showed that carboxylated mucopolysaccharides may also be present in cortical granules. The natural ionic environment of these eggs would favor the formation of very stable complexes between sulfated mucopolysaccharides and quaternary ammonium salts. Brief exposure of unfertilized sea urchin eggs to several quaternary ammonium compounds produced a residual adverse effect on subsequent fertilization in terms of increased vulnerability to polyspermy and reduced fertilizability. These results suggest that sulfated acid mucopolysaccharides participate in the function of the cortical granules and the establishment of the block to polyspermy at fertilization, and possibly in other cellular secretory processes.     

An Improved Silver Stain for Developing Nervous Tissue

A reduced silver technique using physical development to stain embryonic nervous tissue is described. Brains are fixed in Bodian's fixative. Paraffin sections are pretreated with 1% chromic acid or 5% formol. They are impregnated with 0.01% silver nitrate dissolved in 0.1 M boric acid/sodium tetraborate buffer of pH 8 or with silver proteinate. Finally they are developed in a special physical developer which contains 0.1% silver nitrate, 0.01-0.l% formol as developed agent, 25% sodium carbonate to buffer the solution at pH 10.3, 0.1% ammonium nitrate to prevent precipitation of silver hydroxide, and 5% tungstosilicic acid as a protective colloid. The development takes several minutes in this solution, thus the intensity of staining can be controlled easily. The method yields uniform, complete and reproducible staining of axons at all developmental stages of the nervous tissue and is easy to handle.     

An improved silver stain for developing nervous tissue.

A reduced silver technique using physical development to stain embryonic nervous tissue is described. Brains are fixed in Bodian's fixative. Paraffin sections are pretreated with 1% chromic acid or 5% formol. They are impregnated with 0.01% silver nitrate dissolved in 0.1 M boric acid/sodium tetraborate buffer of pH 8 or with silver proteinate. Finally they are developed in a special physical developer which contains 0.1% silver nitrate, 0.01-0.1% formol as reducing agent, 2.5% sodium carbonate to buffer the solution at pH 10.3, 0.1% ammonium nitrate to prevent precipitation of silver hydroxide, and 5% tungstosilicic acid as a protective colloid. The development takes several minutes in this solution, thus the intensity of staining can be controlled easily. The method yields uniform, complete and reproducible staining of axons at all developmental stages of the nervous tissue and is easy to handle.     

Electron microscopic studies of lipopolysaccharides from phase I and phase II Coxiella burnetii

Lipopolysaccharides from phase I (LPSI) Coxiella burnetii Ohio and Nine Mile strains and from phase II (LPSII) Nine Mile stain were negatively and positively and examined with the electron microscope. The ultrastructure of LPSI and LPSII positively stained with uranyl formate or uranyl acetate was ribbon-like. When negatively stained with uranyl acetate, LPSI was ribbon-like but LPSII exhibited hexagonal lattice structures. However, LPSII stained negatively with sodium phosphotungstate and ammonium molybdate exhibited hexagonal lattice ultrastructures which were not identical to those observed when negatively stained with uranyl acetate. The hexagonal lattice structures formed in vitro were due to the interactions of LPSII and the staining reagents rather than to protein-LPS interactions. The differences in the ultrastructures of LPSI and LPSII are undoubtedly based on variations in their chemical composition.     

The loss of enzyme reaction products from ultrathin sections during the staining for electron microscopy

Summary The effects of heavy metal salt staining procedures on the reaction products obtained in the demonstration of arylsulphatase and of acid phosphatase were studied.Lead citrate staining at pH 12 was found to cause a very marked dissolution of barium sulphate and a moderate dissolution of lead sulphate. The staining with uranyl acetate was found to dissolve moderately both barium and lead sulphate.Neither lead citrate nor uranyl acetate staining had any remarkable effect on lead phosphate.The mechanism of the dissolution and the possibilities to avoid it were discussed.     

Addition of U(VI) to a uranium mining waste sample and resulting changes in the indigenous bacterial community

Based on 16S rRNA gene sequence retrieval, changes in natural bacterial community structure induced by addition of uranyl or sodium nitrate to soil samples from a uranium mining waste pile were investigated. Our results demonstrate that both treatments cause drastic changes in the bacterial composition of the studied samples, resulting in strongly reducing the originally predominant Acidobacteria and Alphaproteobacteria. The addition of sodium nitrate induced a strong propagation of particular denitrifying and nitrate‐reducing populations belonging to Actinobacteria and Bacteroidetes. The treatment of the samples with uranyl nitrate demonstrated that most part of the mentioned Bacteroidetes and some of the actinobacterial populations do not tolerate high U(VI) concentrations. Instead, a strong propagation of Pseudomonas spp. from the Gammaproteobacteria occurred. At the initial stages of incubation (4 weeks after the addition of uranyl nitrate) U(VI)‐reducing Geobacter spp. appeared. However, at the later stages of incubation (14 weeks after the beginning of supplementation) no Geobacter populations were detected anymore. Interestingly, different U‐sensitive Bacteroidetes and alphaproteobacterial populations propagated in the U(VI)‐treated samples at these late stages of incubation. That indicated that the added U(VI) was no longer bioavailable. The drastic changes in bacterial community structure of the soil samples from the depleted uranium mining waste caused by the addition of uranyl nitrate indicate that most of the established indigenous bacterial populations do not tolerate U(VI). By the treatment with uranyl nitrate they are replaced by particular uranium resistant nitrate‐reducing and denitrifying populations that potentially interact with the added radionuclide. On the other hand, the large number of dead uranium‐sensitive bacteria likely liberates phosphate‐rich and other biopolymers capable of binding U(VI). On the basis of our results, we propose that bacteria along with the abiotic soil components such as minerals and humic acids may influence the behaviour of U(VI) in nature.     

Electron Dense Artefactual Deposits in Tissue Sections: The Role of Ethanol, Uranyl Acetate and Phosphate Buffer

The occurrence of electron dense deposits in sections of aldehyde-fixed tissue prepared for transmission electron microscopy has been attributed to a number of conflicting factors. In an attempt to clarify this, the precipitating effect of different combinations of phosphate or cacodylate buffer, glutaraldehyde, ethanol and uranyl acetate was investigated in test tubes. As a preliminary investigation the combination of phosphate buffer, ethanol and uranyl acetate was investigated in heart and kidney tissue fixed in glutaraldehyde with or without postosmication. The essential factors in the formation of electron dense deposits in these tissues appear to be phosphate buffer, ethanol, and uranyl acetate, although glutaraldehyde may contribute in some way. The nature and intensity of the deposits seem to vary with the sequence of combination of these factors. Osmium did not appear to be an essential factor in the reaction since deposits were observed in both osmicated and unosmicated tissue. To avoid such deposits, a postosmication distilled water wash for 20 to 30 min followed by en bloc staining with aqueous uranyl acetate is advised if phosphate buffer is used as a fixative vehicle or buffer wash after the primary fixative.     

Electron dense artefactual deposits in tissue sections: the role of ethanol, uranyl acetate and phosphate buffer.

The occurrence of electron dense deposits in sections of aldehyde-fixed tissue prepared for transmission electron microscopy has been attributed to a number of conflicting factors. In an attempt to clarify this, the precipitating effect of different combinations of phosphate or cacodylate buffer, glutaraldehyde, ethanol and uranyl acetate was investigated in test tubes. As a preliminary investigation the combination of phosphate buffer, ethanol and uranyl acetate was investigated in heart and kidney tissue fixed in glutaraldehyde with or without postosmication. The essential factors in the formation of electron dense deposits in these tissues appear to be phosphate buffer, ethanol, and uranyl acetate, although glutaraldehyde may contribute in some way. The nature and intensity of the deposits seem to vary with the sequence of combination of these factors. Osmium did not appear to be an essential factor in the reaction since deposits were observed in both osmicated and unosmicated tissue. To avoid such deposits, a postosmication distilled water wash for 20 to 30 min followed by en bloc staining with aqueous uranyl acetate is advised if phosphate buffer is used as a fixative vehicle or buffer wash after the primary fixative.     

The correlation between bismuth and uranyl staining and phosphorus content of intracellular structures as determined by electron spectroscopic imaging

Four groups of intracellular structures can be recognized according to bismuth and uranyl staining and phosphorus content. (1) Those which contain phosphorus and stain strongly with uranyl acetate but not with bismuth (ribosomes, heterochromatin and mature ribosomal precursor granules), presumably because of their nucleic acid content. (2) Those which contain phosphorus and stain with uranyl acetate and bismuth (interchromatin granules, immature ribosomal precursor granules and mitochondrial granules), presumably because at least some of their phosphate is available to react with bismuth. (3) Those which contain little phosphorus but which stain strongly with bismuth and weakly with uranyl acetate (Golgi complex beads), perhaps because some ligand in addition to phosphate reacts with bismuth, and (4) those which do not contain phosphorus and stain with neither uranyl acetate nor bismuth (portasomes). Uranyl staining correlates strongly with the phosphorus content of nucleic acids, proteins and inorganic deposits. Bismuth will stain some phosphorylated molecules but not all. Thus only some phosphates stain with bismuth.     

An alternative Alcian Blue dye variant for the evaluation of fetal cartilage

BACKGROUND: The most comprehensive evaluation of vertebrate skeletal development involves the use of Alizarin Red S dye to stain ossified bone and various other dyes to stain cartilage. The dye used most widely to stain fetal cartilage in rodents and rabbits is Alcian Blue 8GX. However, the global supply of this specific dye has been exhausted. Several forms of the dye marketed as Alcian Blue 8GX are now available, although they are not synthesized via the original 8GX manufacturing process. METHODS: One new Alcian Blue 8GX form and two Alcian Blue dye variants were evaluated in rats and rabbits using standard staining procedures. The staining quality of these dyes were evaluated relative to the original form of Alcian Blue 8GX based on cartilage uptake of the dye, clarity of the cartilaginous components, staining intensity of the dye, and overall readability of the specimens under stereomicroscopic evaluation. RESULTS: Staining with the newer form of Alcian Blue 8GX resulted in poor staining quality. The Alcian Blue-Pyridine variant performed well, although staining intensity was less than optimal. The Alcian Blue-Tetrakis variant provided staining characteristics that were most similar to the original form of Alcian Blue 8GX. CONCLUSIONS: Alcian Blue-Tetrakis was markedly better in its ability to stain fetal cartilage than the newer form of Alcian Blue 8GX.     

Effects of inorganic nitrogen sources on the production of PP-V [(10Z)-12-carboxyl-monascorubramine] and the Expression of the nitrate assimilation gene cluster by Penicillium sp. AZ

A fungal strain, Penicillium sp. AZ, produced the azaphilone Monascus pigment homolog when cultured in a medium composed of soluble starch, ammonium nitrate, yeast extract, and citrate buffer, pH 5.0. One of the typical features of violet pigment PP-V [(10Z)-12-carboxyl-monascorubramine] is that pyranoid oxygen is replaced with nitrogen. In this study, we found that ammonia and nitrate nitrogen are available for PP-V biosynthesis, and that ammonia nitrogen was much more effective than nitrate nitrogen. Further, we isolated nitrate assimilation gene cluster, niaD, niiA, and crnA, and analyzed the expression of these genes. The expression levels of all these genes increased with sodium nitrate addition to the culture medium. The results obtained here strongly suggest that Penicillium sp. AZ produced PP-V using nitrate in the form of ammonium reduced from nitrate through a bioprocess assimilatory reaction.     

Comparative ultrastructural study on tris 1-aziridinyl phosphine oxide prefixed cuticles of some oligochaetes

The cuticle of five species of Oligochaeta, chosen to represent differences in size and a variety of biotopes, was studied electron microscopically after fixation with the acrolein-TAPO-osmium tetroxide method. Five distinct layers in the cuticle of all studied species were found. Staining with lead and uranyl ions or with silver proteinate visualized basically the same structural components of the cuticle, but the degree of electron opacity and the distribution of the electron-opaque stain in these components differed according to the staining method used. Since the acrolein-TAPO-osmium tetroxide method visualized the cuticular zones preferentially stained by Thiéry's silver proteinate method, it was concluded that the TAPO method may be considered suitable for the visualization of polysaccharides. Staining with phosphotungstic acid provided some information on the composition of the cuticle of Oligochaeta not obtained by staining ultrathin sections with lead and uranyl ions nor with silver proteinate. The conclusion is that phosphotungstic acid binds to polysaccharides which do not contain vicglycol groups nor active sites responsible for the positive reaction with lead and uranyl salts. Structural components in the cuticle of the oligochaetes studied were characteristic for each species. The taxonomic value of such components, however, must be confirmed by examination of a larger number of species of oligochaetes.     

Ultrastructural staining with sodium metaperiodate and sodium borohydride.

This article describes new ultrastructural staining methods for osmicated tissues based on the incubation of sections with sodium metaperiodate and sodium borohydride solutions before uranyl/lead staining. Sections incubated with sodium metaperiodate and sodium borohydride, treated with Triton X-100, and stained with ethanolic uranyl acetate/lead citrate showed a good contrast for the nucleolus and the interchromatin region, whereas the chromatin masses were bleached. Chromatin bleaching depended on the incubation with these oxidizing (metaperiodate) and reducing (borohydride) agents. Other factors that influenced the staining of the chromatin masses were the en bloc staining with uranyl acetate, the incubation of sections with Triton X-100, and the staining with aqueous or ethanolic uranyl acetate. The combination of these factors on sections treated with metaperiodate/borohydride provided a different appearance to the chromatin, from bleached to highly contrasted. Most cytoplasmic organelles showed a similar appearance with these procedures than with conventional uranyl/lead staining. However, when sections were incubated with metaperiodate/borohydride and Triton X-100 before uranyl/lead staining, the collagen fibers, and the glycocalix and zymogen granules of pancreatic acinar cells, appeared bleached. The possible combination of these methods with the immunolocalization of the amino acid taurine was also analyzed. (J Histochem Cytochem 50:11-19, 2002)     

ELECTRON MICROSCOPY OF DNA MOLECULES "STAINED" WITH HEAVY METAL SALTS

Single DNA molecules can be rendered visible in the electron microscope by "staining" with water-soluble salts of heavy metals. The best results were obtained with lanthanum nitrate, uranyl acetate, and lead perchlorate. The molecules appear as filaments approximately 20 A wide. Their length was not determined, but it could be shown that it varied with the molecular weight of the DNA used. The same heavy metal salts will preferentially "stain" the nucleic acid in a protein-DNA complex. Evidence is provided for the possibility of a partial separation of a double-stranded molecule into single strands on adsorption to the supporting film.     

Use of Hydrogen Peroxide to Accelerate Staining of Ultrathin Spurr Sections

Pretreatment of ultrathin Spurr sections of glutaraldehyde-osmium fixed tissue with hydrogen peroxide significantly reduces the time required to stain with ethanolic uranyl acetate and lead citrate for electron microscopy. Micrographs compare contrast obtained by this method with that obtained using conventional staining times. Reasons for the facilitating action of hydrogen peroxide are suggested.     

Simultaneous demonstration of glycogen and protein in glycosomes of cardiac tissue

Cardiac conduction fibers fixed either in glutaraldehyde and OsO4 or treated additionally en bloc with uranyl acetate were studied in order to demonstrate the structure of glycosomes (protein-glycogen complex). Sections were stained histochemically by periodic acid-thiosemicarbazide-silver proteinate (PA--TSC--SP) for glycogen followed by uranyl acetate and lead citrate (U-Pb) for protein. In control sections periodic acid was replaced by hydrogen peroxide (H2O2). Glycogen appeared in all sections stained by PA-TSC-SP. Protein was poorly contrasted in periodic acid treated histochemical sections taken from fixed in glutaraldehyde and OsO4. Simultaneous staining of glycogen and protein was achieved in sections of tissue treated en bloc with uranyl acetate. This treatment revealed two classes of glycosomes: 1) glycosomes deposited freely in the cytoplasm whose structure was disintegrated after treatment with uranyl acetate: 2) glycosomes associated with other cellular structures that remained intact. Staining of glycogen and protein in the same section demonstrated for the first time the structure of intact glycosomes.