Alkaline bismuth solution as an en bloc stain for formaldehyde-glutaraldehyde potassium permanganate fixed fungal spores

An alkaline solution of bismuth subnitrate reacted well with the cell membranes and cell walls of formaldehyde-glutaraldehyde potassium permanganate fixed Alternaria spores, demonstrating them with greater contrast than in sections stained with uranyl acetate and lead citrate. Optimal fine structure of fungal spores was obtained by en bloc staining with alkaline bismuth solution after aldehyde and permanganate fixation. The contrast of the cell organelles and cell walls was high enough in sections cut after the alkaline bismuth en bloc stain for direct ultrastructural observation. Our results indicate that the alkaline bismuth stain is useful either as an en bloc or section stain for aldehyde and permanganate fixed fungal spores.     

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.     

High-contrast en bloc staining of neuronal tissue for field emission scanning electron microscopy

Conventional heavy metal poststaining methods on thin sections lend contrast but often cause contamination. To avoid this problem, we tested several en bloc staining techniques to contrast tissue in serial sections mounted on solid substrates for examination by field emission scanning electron microscopy (FESEM). Because FESEM section imaging requires that specimens have higher contrast and greater electrical conductivity than transmission electron microscopy (TEM) samples, our technique uses osmium impregnation (OTO) to make the samples conductive while heavily staining membranes for segmentation studies. Combining this step with other classic heavy metal en bloc stains, including uranyl acetate (UA), lead aspartate, copper sulfate and lead citrate, produced clean, highly contrasted TEM and scanning electron microscopy (SEM) samples of insect, fish and mammalian nervous systems. This protocol takes 7-15 d to prepare resin-embedded tissue, cut sections and produce serial section images.     

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.     

Lead staining in the Golgi complex

Lead ions at similar concentrations to those used for Gomori type phosphatase localization stain some parts of the vacuolar system, particularly compartments of the Golgi complex (GC) and isolation envelopes (im) in a characteristic way in both vertebrates and invertebrates. After fixation in 2.5% glutaraldehyde, lead citrate in acetate or aspartate buffer (pH 5.5-7.2) leaves the contents of GC cisternal compartments with a fine particulate stippling. In the fat body of Calpodes ethlius and in mouse pancreas the staining is faint but definite without further enhancement of contrast, although it is easily overlooked after section staining. The distribution of lead stain differs from that of the lead phosphate precipitated after Gomori type acid phosphatase reactions. Whereas lead stain may be in all GC and im compartments, acid phosphatase is restricted to the innermost saccules and nearby vacuoles. The compartment specific staining by led also differs from the generalized staining in all compartments given by uranyl. Thus the contents of luminal membrane surfaces of some parts of the vacuolar system can be characterized by their ability to bind lead. In cells where protein synthesis has been blocked by cycloheximide, secretory vesicles are absent and the RER and GC from the generalized staining in all compartments given by uranyl. Thus the contents of luminal membrane surfaces of some parts of the vacuolar system can be characterized by their ability to bind lead. In cells where protein synthesis has been blocked by cycloheximide, secretory vesicles are absent and the RER and GC from the generalized staining in all compartments given by uranyl. Thus the contents of luminal membrane surfaces of some parts of the vacuolar system can be characterized by their ability to bind lead. In cells where protein synthesis has been blocked by cycloheximide, secretory vesicles are absent and the RER and GC cisternae are devoid of uranyl stainable material. However, lead staining and acid phosphatase activity in the GC continue. We presume that they mark the environment within these cisternae rather than the proteins passing through them. This environment is itself not static. Several observations suggest that the function of cisternae that is detectable by lead staining is temporally discontinuous and related to a stage of maturation or development. Only early stage ims stain: the staining ceases by the beginning of autophagy after hydrolytic enzymes are presumed to have been added. Condensing vacuoles cease to stain as the central core crystallizes out. Stain may be absent from one or two GC saccules at any position in the stack as though the phase of lead staining (or lack or it) can move progressively through the system. We conclude that in studies characterizing components of the vacuolar system it is necessary to separate those that mark transient occupants of a compartment from those that mark the compartment itself. Both may vary temporally independently from one another.     

Alkaline Bismuth Solution as an En Bloc Stain for Formaldehyde-Glutaraldehyde Potassium Permanganate Fixed Fungal Spores

An alkaline solution of bismuth subnitrate reacted well with the cell membranes and cell walls of formaldehyde-glutaraldehyde potassium permanganate fixed Alternaria spores, demonstrating them with greater contrast than in sections stained with uranyl acetate and lead citrate. Optimal fine structure of fungal spores was obtained by en bloc staining with alkaline bismuth solution after aldehyde and permanganate fixation. The contrast of the cell organelles and cell walls was high enough in sections cut after the alkaline bismuth en bloc stain for direct ultrastructural observation. Our results indicate that the alkaline bismuth stain is useful either as an en bloc or section stain for aldehyde and permanganate fixed fungal spores.     

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)     

Cytochemical Staining of Sections from Plastic-Embedded Flagellates

Dinoflagellate chromosomes in sections of plastic-embedded cells were stained without removing the plastic. Azur B and Feulgen procedures were used to localise DNA. Azur B was used with Araldite or methacrylate sections by staining in 0.2% stain in 0.05 M citrate buffer at pH 4 for 1 hr at 50 C followed by rinsing in tertiary butyl alcohol to differentiate the chromosomes. Feulgen stain was used with Araldite sections by hydrolyzing in 1 N HCl at 60 C for 10 min, rinsing in water, staining for 24 hr, washing well, drying and covering. Fast green was used with methacrylate sections to stain proteins by flooding the slide with a 0.1% solution of stain in 0.06 M phosphate buffer at pH 8, allowing the stain to dry out at 40-50 C, washing well, drying and covering. Controls were carried out on material fixed in formalin and treated with nucleases or proteolytic enzymes prior to embedding, and staining.     

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/ethylenediaminetetraacetic 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.     

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.     

Ultrastructural silver enhancement of Prussian blue-reactive iron in hematopoietic and intestinal cells

Prussian blue has been widely used to localize iron in a variety of tissues at the light and electron microscopic level. In the present study, thin sections of human marrow and blood cells and rat duodenal cells were exposed to silver proteinate (SP) after staining en bloc with acid ferrocyanide (AF), with and without prior iron saturation using iron nitrilotriacetate (FeNTA). Silver deposition was observed over Prussian blue-reactive sites and significantly enhanced sites of minimal AF and FeNTA-AF staining. AF-SP stain deposits were present in the cytoplasmic matrix, granules, and occasionally on the surfaces of macrophages, monocytes, and erythroblasts. FeNTA-AF-SP stained additional cytoplasmic and surface sites in erythroblasts and stained neutrophil granules intensely. Duodenal epithelium from iron-loaded rats demonstrated strong AF-SP staining of ferric iron in microvilli, apical cytoplasmic matrix, and lateral membranes. Similar preparations from iron-replete rats stained sparsely; however, intense AF-SP staining was observed after iron saturation with FeNTA. SP similarly enhanced luminal ferrous iron deposits stained with acid ferricyanide in rats given intraluminal ferrous iron. AF-SP stain deposits were removed by exposure of thin sections to NH4OH, KCN, or HNO3 but were not affected by prior exposure to HIO4 or NaBH4, consistent with a silver cyanide or complex stain precipitate rather than reduced silver or silver ferriferrocyanide. SP enhancement of Prussian blue allows identification of reactive sites not readily visualized with AF or FeNTA-AF alone, and offers the potential for differentiating AF staining from other deposits or organelles of comparable density.     

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 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.     

LANTHANUM STAINING OF THE SURFACE COAT OF CELLS : Its Enhancement by the Use of Fixatives Containing Alcian Blue or Cetylpyridinium Chloride

Among the techniques which have been reported to stain the surface coat of cells, for electron microscopy, is lanthanum staining en bloc. Similarly, the presence of the cationic dye, Alcian blue 8GX, in a primary glutaraldehyde fixative has been reported to improve the preservation of the surface coat of cells of many types; however, the preserved coat is not very electron opaque unless thin sections are counterstained. The present paper shows that for several rat tissues lanthanum staining en bloc is an effective electron stain for the cell surface, giving excellent contrast, if combined sequentially with prefixation in an aldehyde fixative containing Alcian blue. The cationic substance cetylpyridinium chloride was found to have a similar effect to that of Alcian blue in enhancing the lanthanum staining of the surface coat material of the brush border of intestinal epithelial cells. The patterns of lanthanum staining obtained for the tissues studied strikingly resemble those reported in the literature where tissues are stained by several standard methods for demonstrating mucosubstances at the ultrastructural level. This fact and the reproduction of the effect of Alcian blue by cetylpyridinium chloride constitute a persuasive empirical argument that the material visualized is a mucopolysaccharide or mucopolysaccharide-protein complex.     

Cytochemical Staining of Sections from Plastic-Embedded Flagellates

Dinoflagellate chromosomes in sections of plastic-embedded cells were stained without removing the plastic. Azur B and Feulgen procedures were used to localise DNA. Azur B was used with Araldite or methacrylate sections by staining in 0.2% stain in 0.05 M citrate buffer at pH 4 for 1 hr at 50 C followed by rinsing in tertiary butyl alcohol to differentiate the chromosomes. Feulgen stain was used with Araldite sections by hydrolyzing in 1 N HCl at 60 C for 10 min, rinsing in water, staining for 24 hr, washing well, drying and covering. Fast green was used with methacrylate sections to stain proteins by flooding the slide with a 0.1% solution of stain in 0.06 M phosphate buffer at pH 8, allowing the stain to dry out at 40-50 C, washing well, drying and covering. Controls were carried out on material fixed in formalin and treated with nucleases or proteolytic enzymes prior to embedding, and staining.     

Observations on the kinetics of uranyl acetate and phosphotungstic acid staining of chromatin in thin sections for electron microscopy

When thin sections of spermatogenic chromatin are fixed with either glutaraldehyde alone or postfixed with osmium tetroxide (OsO4) and stained with uranyl acetate (UAc) for increasing times, even after as little as 1 min, stain uptake is proportional to section thickness. Greater UAc uptake is observed in chromatin fixed with glutaraldehyde only, but seen with postfixed chromatin. Lead citrate poststaining of chromatin fixed with either glutaraldehyde or postfixed with OsO4 increases UAc uptake by a factor of about 3. The staining of thin sections of spermatogenic chromatin with ethanolic phosphotungstic acid (PTA) shows a region where stain uptake is proportional to section thickness followed by a plateau. This staining pattern is seen in chromatin fixed with glutaraldehyde alone or postfixed with OsO4; similar levels for final PTA uptake are also observed. An increase in the resin content of embedded chromatin postfixed with OsO4 is proposed to explain the decrease and increase in the rate of migration of UAc and ethanolic PTA staining solutions, respectively.     

A Method for Visualizing the Acrosome by Light Microscopy

A silver staining technique applied to squash preparations of material previously fixed in 3:1 ethanol: acetic acid produces differential staining of the acrosomal region of spermatids during spermiogenesis in orthopteroid species. The method includes treatment with saline sodium citrate solution for 15 min at 60 C, and staining with 50% aqueous silver nitrate adjusted to pH 2.9 with formic acid.     

Rapid primary microwave-osmium fixation. I. Preservation of structure for electron microscopy in seconds

Microwave irradiation (MWIr) of tissues immersed in aldehydes has been used to preserve fine structure in seconds. The purpose of this study was to extend these findings to include rapid primary osmium fixation in a microwave (MW) device with a high volume exhaust. Blocks of rat heart and liver were trimmed to approximately 4 mm3 and exposed to 0.2 M symcollidine-buffered 2% osmium tetroxide for a period of 6-7 sec during MWIr (final solution temperature approximately 45 degrees C). We also evaluated rapid fixation of tissues exposed to MWIr simultaneously with immersion in dilute Karnovsky's fixative (6-7 sec to approximately 50 degrees C) followed by MWIr of specimens immersed in osmium (7 sec to approximately 45 degrees C). Tissues were stored in 0.1 M sodium cacodylate buffer (pH 7.3, 4 degrees C) up to 2 weeks and were stained en bloc in uranyl acetate, dehydrated in a graded series of alcohols, and embedded in propylene oxide-Epon sequence. Thin sections were stained with lead citrate and examined by transmission electron microscopy. We demonstrate that fine structural preservation of tissue blocks can be achieved by MWIr in aldehyde and/or osmium in seconds.     

A method for visualizing the acrosome by light microscopy

A silver staining technique applied to squash preparations of material previously fixed in 3:1 ethanol:acetic acid produces differential staining of the acrosomal region of spermatids during spermiogenesis in orthopteroid species. The method includes treatment with saline sodium citrate solution for 15 min at 60 C, and staining with 50% aqueous silver nitrate adjusted to pH 2.9 with formic acid.