Chromoxane cyanine R. II. Staining of animal tissues by the dye and its iron complexes

The staining properties of chromoxane cyanine R (Colour Index No. 43820, Mordant blue 3; also known as eriochrome cyanine R and solochrome cyanine R) have been studied. Used alone, the dye imparted its red colour to nuclei, cytoplasm and collagen. The dye was extracted by mild alkali but not by acids. Stainability required ionized amino groups in the tissue, and there was also evidence for non-ionic binding of the dye. The colours obtained by staining with mixtures of chromoxane cyanine R and ferric chloride varied with the molar iron: dye ratio and with the pH. Useful staining was seen only between pH 1 and 2. The tissues were coloured either all blue (when Fe: dye was high), or both red and blue (when Fe: dye was low). Lower pH favoured the deposition of red, higher pH the deposition of blue colour. The red was mainly in cytoplasm, blue in nuclei and myelin. Collagen fibres were red or purple, depending on pH and iron: dye ratio. Red colours were differentiated by acid and changed to blue, but not extracted, by mild alkali. The red substance in the stained sections was clearly not the free dye, so it was probably an iron-dye complex. From the effects of various differentiating agents, it was deduced that the red and blue dye-metal complex molecules were bound to the tissue by the dye moiety, not by interposition of iron atoms. Staining by the complexes of iron(III) with chromoxane cyanine R did not involve nucleic acids or other polyanions or the amino groups of proteins. There was evidence for only non-ionic binding of both red and blue complexes. It is suggested that the red colour in sections stained by solutions with low iron: dye ratio is due to a simple carboxylate complex, [Fe₂H(dye)]^?. The blue colour would then result from withdrawal of a proton from the red complex to give [Fe₂(dye)]^2-. The bases that remove the protons may be arginine-rich nucleoproteins of nuclei and phospholipid bases of myelin. Techniques are described for informative simultaneous staining in two colours, and for the selective staining of either nuclei or myelin.     

A new technique for obtaining large platinum-carbon replicas

A problem often encountered in freeze-fracturing is that platinum-carbon replicas roll up or are broken into fragments during tissue digestion and replica washing. In replicas damaged in these ways, tissue orientation and cell identification are difficult. In order to prevent such damage, methods have been introduced of replica strengthening by coating them with plastic films (Steere, 1957; Willison & Rowe, 1980; Stolinski et al., 1983), or with an additional layer of silver or gold (Robards & Umrath, 1978; Robards & Sleytr, 1985). A disadvantage of these methods, however, is that the additional layer must be removed before the replicas can be examined under the electron microscope. Removal of the additional layer results in loss of image quality. A reinforcing plastic film may not be completely digested by the tissue solvent and will then contaminate the replica, while the silver technique requires a silver evaporation source to be present in the vacuum chamber. Instead of replica strengthening methods, some authors recommend putting replicas carrying tissues, which have been partially digested by bleach (Pearce, 1983) or chromic acid (Fetter & Costello, 1986), onto carbon-coated gold grids and then floating them on chromic acid. However, neither this method nor the replica strengthening method assure the production of large unrolled replicas. Moreover, gold grids are rather expensive.     

Fungal mediated synthesis of silver nanoparticles and evaluation of antibacterial activity

Nanoparticles as biomedicine has made a crucial role in health biotechnology. Different transition metals in various forms playing role in nanotechnological advances and biological applications. Silver as one of the nontoxic, safe inorganic antibacterial agents and can serve as replacement of antibiotics. Present research is based on biogenic synthesis of silver nanoparticles (Ag‐NPs) as potential antibiotics from fungal metabolites of Penicillium oxalicum. We used different analytical techniques X‐ray diffraction (XRD) and scanning electron microscopy (SEM) for characterization of biosynthesized silver nanoparticles. Furthermore, the antibacterial activity of biosynthesized silver nanoparticles was checked against Staphylococcus aureus, S. dysenteriae, and Salmonella typhi by using well diffusion method and UV visible spectrophotometer. Maximum zone of inhibition recorded against S. aureus, Shigella dysenteriae was 17.5 ± 0.5 mm (mm) for both species and 18.3 ± 0.60 mm for Salmonella typhi. The biosynthesized silver nanoparticles of P. oxalicum showed excellent antibacterial activity. It was concluded from our results that biosynthesized silver nanoparticles have significant potential and might be useful for a wide range of biological applications such as bactericidal agent against resistant bacteria, preventing infections, healing wounds, and anti‐inflammation.     

Antimicrobial effect,frictional resistance,and surface roughness of stainless steel orthodontic brackets coated with nanofilms of silver and titanium oxide: a preliminary study

Nano‐silver and nano‐titanium oxide films can be coated over brackets in order to reduce bacterial aggregation and friction. However, their antimicrobial efficacy, surface roughness, and frictional resistance are not assessed before. Fifty‐five stainless‐steel brackets were divided into 5 groups of 11 brackets each: uncoated brackets, brackets coated with 60 µm silver, 100 µm silver, 60 µm titanium, and 100 µm titanium. Coating was performed using physical vapor deposition method. For friction test, three brackets from each group were randomly selected and tested. For scanning electron microscopy and atomic‐force microscopy assessments, one and one brackets were selected from each group. For antibacterial assessment, six brackets were selected from each group. Of them, three were immediately subjected to direct contact with S. mutans. Colonies were counted 3, 6, 24, and 48 h of contact. The other three were stored in water for 3 months. Then were subjected to a similar direct contact test. Results pertaining to both subgroups were combined. Groups were compared statistically. Mean (SD) friction values of the groups 'control, silver‐60, silver‐100, titanium‐60, and titanium‐100' were 0.55 ± 0.14, 0.77 ± 0.08, 0.82 ± 0.11, 1.52 ± 0.24, and 1.57 ± 0.41 N, respectively (p = .0004, Kruskal–Wallis). Titanium frictions were significantly greater than control (p < .05), but silver groups were not (p > .05, Dunn). In the uncoated group, colony count increased exponentially within 48 h. The coated groups showed significant reductions in colony count (p < .05, two‐way‐repeated‐measures ANOVA). In conclusions, all four explained coatings reduce surface roughness and bacterial growth. Nano‐titanium films are not suitable for friction reduction. Nano‐silver results were not conclusive and need future larger studies.     

Accumulation of fluorescent non-cationic probes in mitochondria of cultured cells: observations,a proposed mechanism,and some implications

Cultured rat fibroblasts were exposed to millimolar concentrations of forty-four non-cationic fluorescent probes, of very varied physico-chemical properties. Mitochondrial staining occurred with nineteen of these probes, nine of which were nominally anionic and ten nominally non-ionic. All nineteen were in fact lipophilic weak acids. Using structural parameters these could be specified numerically as follows: electric charge ≤ 0; log P(less-ionized form) < 0; and pKa ～ 7. In addition to these structural variables, dye concentration and the time of exposure of cells to probes were significant factors for the staining of mitochondria. Accumulation of these compounds can be understood in terms of ion-trapping of hydrophilic salts of lipophilic weak acids, due to the internal pH of respiring mitochondria being higher than the cytosolic pH. As a case example of the application of this approach, the mode of action of many inhibitors of mitochondrial anabolism is discussed in terms of the mechanisms introduced here.     

Nanoleakage evaluation of resin luting systems to dental enamel and leucite-reinforced ceramic

Purpose: The aim of this study was to evaluate the nanoleakage patterns between dental enamel and reinforced leucite ceramic, bonded with resin luting systems and a flowable composite resin. Materials and Methods: Twelve crowns of bovine incisors were randomly divided into four groups (n = 3) according to the luting procedure: Excite/Variolink II, Clearfil SE Bond/Panavia F, Scotchbond Multi‐Purpose Plus/RelyX ARC, and Single Bond 2/Filtek Z350 Flow. To evaluate the nanoleakage patterns, IPS Empress Esthetic disks (5 mm Ø and 1.2‐mm thick) were bonded to enamel, and, after 24 h, the specimens were immersed in a 50% (w/v) solution of silver nitrate (24 h), fixed, dehydrated, and processed scanning electron microscopy (SEM). Results: None nanoleakage on interface of the groups that Single Bond 2 followed by the flowable composite were used. The highest percentage of nanoleakage was shown by the Excite/Variolink II protocol. Also, in all conditions tested, none silver nitrate uptake was observed between the leucite‐reinforced ceramic and the resin luting cement. Conclusions: The use of a two‐step etch‐and‐rinse adhesive with flowable composite was able to promote an adequate seal of the bond interface at the enamel. Moreover, the conventional dual‐cured resin cements associated with simplified and dual‐cured adhesives tested are also indicated to bond thin ceramics to enamel, since all presented low silver nitrate uptake. Microsc. Res. Tech., 2012. © 2011 Wiley Periodicals, Inc.     

A method for abolishing the nonspecific binding artifacts produced by the peroxidase-antiperoxidase complex (PAP) in immunostained mucous epithelia

The presence of nonspecific staining artifacts is a potential problem in ultrastructural immunocytochemistry. In the course of staining for lysozyme in the human and rabbit endocervix, the peroxidase-antiperoxidase complex (PAP) was found to bind to mucous granules in a selective and nonspecific manner. Sections etched in 10% aqueous H₂O₂, incubated for 5 minutes in 1:50 PAP in Tris-buffered saline (TBS) and subsequently treated with 3, 3′-diaminobenzidine-H₂O₂, revealed a staining precipitate within the matrix of mucous granules. The same selective and nonspecific staining could also be visualized when horseradish peroxidase (1 mg/ml) was substituted for PAP in the immunocytochemical sequence. Thus, this staining method made the reliable demonstration of the desired antigen difficult. The affinity of PAP for mucous granules can be completely eliminated by subjecting sections, previously etched in H₂O₂ to expose the antigenic sites, to a 3-minute incubation in immunoglobulin (1:10 human IgA, 1:5 human IgG, or 1:5 antihuman IgG) immediately prior to using PAP in the standard immunocytochemical staining sequence. The use of this modification to Sternberger's unlabeled antibody-enzyme method is recommended because it allows for the elimination of all nonspecific staining artifacts without interfering with specific localization by the primary antibody. The mechanism that causes nonspecific binding of peroxidase to mucous granule constituents is unclear, although carbohydrate binding may play a role in the interaction between mucous granules and peroxidase.     

Silver enhancement of Nanogold particles during freeze substitution for electron microscopy

Recent advances in rapid freezing and fixation by freeze substitution have allowed structural cell biologists to apply these reliable modes of sample preparation to a wide range of specimens and scientific problems. Progress in electron tomography has produced cellular images with resolution approaching 4 nm in 3D, but our ability to localize macromolecules in these well‐fixed, well‐resolved samples has remained limited. When light fixation and low temperature embedding are employed with appropriate resins, immuno‐localizations can recognize antigens at a section's surface, but labelling is therefore confined, not throughout the section's depth. Small, electron‐dense markers, like Nanogold®, will often enter a living cell, serving as reliable tracers for endocytic activity, but these markers are usually too small to be visible in the context of a cell. We have developed a method for the silver enhancement of Nanogold particles that works during freeze substitution in organic solvents at low temperature. Here, we describe the development of this method, based on in vitro tests of reagents and conditions. We then show results from application of the method to an in vivo system, using Nanogold to track the internalization of immunoglobulin by neonatal murine intestinal epithelium, a specific example of receptor‐mediated membrane traffic.     

Friction and Wear Behaviors of Ni-based Composites Containing Graphite/Ag2MoO4 Lubricants

In order to improve the tribological properties of Ni-based composites, novel adaptive Ni-based composites containing multiple lubricants were prepared via a mechanical alloying and hot-press sintering technique. The phase constituents and microstructure of the composites were characterized and the tribological properties were evaluated from room temperature to 700 °C. The results showed that the Ag₂MoO₄ phase decomposed and new phases of Mo₂C, Ag, and MoO₃ formed in the sintered composites, which can be attributed to the solid state reaction of silver molybdate lubricant during the sintering process. The wear test results indicated that the Ni-based composites containing graphite and silver molybdate lubricants exhibited superior tribological properties at ambient and high temperatures. Subsequently, the Raman results demonstrated that the composition of the tribo-layers on the worn surface of the Ni-based composites was varied with increasing temperature. Combined with the wear test results, it can be proposed that the improvement of tribological properties is due to the synergistic lubricating action of silver molybdate, iron oxide, and nickel oxide. Furthermore, Raman results of the composite containing silver molybdate and silver/molybdenum trioxide lubricants revealed that the silver molybdate lubricant can reproduce easily by the direct reaction between molybdenum trioxide and silver in the agglomerate state.     

Hydrogen peroxide staining to visualize intracellular bacterial infections of seedling root cells

Visualization of bacteria in living plant cells and tissues is often problematic due to lack of stains that pass through living plant cell membranes and selectively stain bacterial cells. In this article, we report the use of 3,3′‐diaminobenzidine tetrachloride (DAB) to stain hydrogen peroxide associated with bacterial invasion of eukaryotic cells. Tissues were counterstained with aniline blue/lactophenol to stain protein in bacterial cells. Using this staining method to visualize intracellular bacterial (Burkholderia gladioli) colonization of seedling roots of switch grass (Panicum virgatum), we compared bacterial free seedling roots and those inoculated with the bacterium. To further assess application of the technique in multiple species of vascular plants, we examined vascular plants for seedling root colonization by naturally occurring seed‐transmitted bacteria. Colonization by bacteria was only observed to occur within epidermal (including root hairs) and cortical cells of root tissues, suggesting that bacteria may not be penetrating deeply into root tissues. DAB/peroxidase with counter stain aniline blue/lactophenol was effective in penetration of root cells to selectively stain bacteria. Furthermore, this stain combination permitted the visualization of the bacterial lysis process. Before any evidence of H₂O₂ staining, intracellular bacteria were seen to stain blue for protein content with aniline blue/lactophenol. After H₂O₂ staining became evident, bacteria were often swollen, without internal staining by aniline blue/lactophenol; this suggests loss of protein content. This staining method was effective for seedling root tissues; however, it was not effective at staining bacteria in shoot tissues due to poor penetration. Microsc. Res. Tech. 77:566–573, 2014. © 2014 Wiley Periodicals, Inc.     

The influence of water vapour in air on the friction behaviour of pure metals during fretting

An investigation was conducted to determine the effect of water vapour content in air on the frictional behaviour during fretting of pure metals: iron, aluminium, copper, silver, chromium, titanium and nickel. The fretting experiments were carried out under various humidity levels, ranging from dry air to 50% relative humidity at 23°C. During the experiment the frictional force between fretting surfaces was measured. Pure metals, except iron, were found to have a maximum value of the coefficient of friction during the steady-fretting stage (μ_s) at a specific humidity (RH_max). Iron showed a rapid decrease in μ_s with increasing humidity at RH_max. Each pure metal also exhibited maximum fretting wear at RH_max. The value of μ_s at RH_max for each metal was strongly related to the heat of formation of the lower metal oxide, indicating that the adhesive contact area was larger at RH_max for the fretting of metals with less chemical activity. At high humidity levels water vapour generally reduced the coefficient of friction, μ_s.     

Histology and ultrastructure of the testis and vas deferens in Pseudochorthippus parallelus parallelus (Orthoptera,Acrididae)

Pseudochorthippus parallelus parallelus (Zetterstedt, 1821) (Orthoptera, Acrididae) is a widespread species in Europe, and also it is localized in some regions in Turkey such as Bursa, Eski?ehir, Ankara, Bolu, Düzce, and Çank?r?. The features of the reproductive organs such as the numbers and shapes of testes and follicles can be used as taxonomical characters. For this purpose, the ultrastructural and histological features of testis and vas deferens in P. parallelus parallelus were examined with using light microscope, scanning electron microscope, and transmission electron microscope. The mature P. parallelus parallelus has two conjugated testes produce spermatozoa. Each testis is composed of numerous testis follicles in which different stages of spermatogenesis and spermiogenesis develop. First, spermatocytes are formed by the mitosis division of the germ cells at the distal end of the follicles. Then, spermatocytes form spermatids by meiosis division in the middle region of the follicles. Finally, spermatids are differentiated to spermatozoa at the proximal region of the follicles. After maturation of the spermatozoa, sperm tails come together as the sperm bundles called as spermatodesm. Each follicle is connected to vas deferens via vas efferens to discharging spermatozoa. In spite of some differences, the testes and the vas deferens in P. parallelus parallelus are highly similar to the those of other species, especially Orthopteran species.     

Staining tissue with methacrylate casts for light microscopy

Scanning electron microscopy (SEM) of methyl methacrylate casts and light microscopy (LM) of tissue are well-established methods for studying the microcirculation. The two are complimentary, but methacrylate is transparent and thus its presence is often not appreciated by LM. Histologic stains applied to methyl methacrylate in tissue sections would better identify by LM and allow the relationships with the SEM view of cast vasculature. We sought to test different stains on cast tissue to find one that would accent the cast. Surgically removed and autopsied human lungs were cast with methacry late and processed by routine light microscopic methods. They were stained with the hematoxylin and eosin, Masson trichome, elastic-van Gieson, Grocottmethenamine silver, Brown-Brennan, and Ziehl-Neelsen methods. The Ziehl-Neelsen procedure stained the methacry late best, giving it a red color. This procedure also worked well without heating. We conclude that (1) cast methacry late lung can be processed for routine LM with excellent results; (2) methacry late stains well with the Ziehl-Neelsen technique; (3) the acid-fast stained cast lung shows capillaries and cells in both normal and diseased lung better than the routine hematoxylin and eosin stain; (4) this technique can be used to assess filling and correlate findings on the same tissue with the two different microscopic methods.     

Microwave-accelerated cytochemical stains for the image analysis and the electron microscopic examination of light microscopy diagnostic slides

Recent studies in our laboratories have shown how microwave (MW) irradiation can accelerate a number of tissue-processing techniques, especially staining, to aid in the preparation of single specimens on glass microscope slides or coverslips for examination by light microscopy (and electron microscopy, if required) for diagnostic purposes. Techniques have been developed, which give permanently stained preparations, that can be studied initially by light microscopy, their areas of interest mapped, and computer-automated image analysis performed to obtain quantitative information. This is readily performed after MW-accelerated staining with silver methenamine by the Giammara-Hanker PATS or PATS-TS reaction. This variation of the PAS reaction gives excellent markers for specific infectious agents such as lipopolysaccharides for gram-negative bacteria or mannans for fungi. It is also an excellent stain for glycogen and basement membranes and an excellent marker for type III collagen or reticulin in the endoneurium or perineurium of peripheral nerve or in the capillary walls. Our improved MW-accelerated Feulgen reaction with silver methenamine for nuclear DNA is useful to show the nuclei of bacteria and fungi as well as of cells they are infecting. Improved coating and penetration of tissue surfaces by thiocarbohydrazide bridging of ruthenium red, applied under MW-acceleration, render biologic specimens sufficiently conductive for SEM so that sputter coating with gold is unnecessary. The specimens treated with these highly visible electron-opaque stains can be screened with the light microscope after mounting in polyethylene glycol(PEG) and the structures or areas selected for EM study are mapped with a Micro-Locator? slide. After removal of the water soluble PEG the specimens are remounted in the usual EM media for scanning electron microscopy (SEM) or transmission electron microscopy (TEM) study of the mapped areas. By comparing duplicate smears from areas of infection, such as two coverslips of buffy coat smears of blood from a patient with septicemia, the microorganisms responsible can occasionally be classified for antimicrobial therapy long before culture results are available; gram-negative bacteria are positive with the Giammara-Hanker PATS-TS stain, and gram-positive bacteria are positive with the SIGMA HT40 Gram stain. The gram-positive as well as gram-negative bacteria are both initially stained by the crystal violet component of the Gram stain. The crystal violet stain is readily removed from the gram-negative (but not the gram-positive) bacteria when the specimens are rinsed with alcohol/acetone. If this rinse step is omitted, the crystal violet remains attached to both gram-negative and gram-positive bacteria. It can then be rendered insoluble, electron-opaque, and conductive by treatment with silver methenamine solution under MW-irradiation. This metallized crystal violet is a more effective silver stain than the PATS-TS stain for a number of gram-negative spirochetes such as Treponema pallidurn, the microbe that causes syphilis     

Some methods for more efficient processing of SEM specimens

Three techniques are described for increasing the quantity of specimens which may be processed for SEM while maintaining the quality of the final product. They are: a simple stainless steel holder for safe manipulation of coverslips during incubation and fixation; a coverslip carrier which permits four or more coverslips to be dehydrated and critically point dried at the same time; and a simple pattern for making baskets to hold relatively large but delicate specimens during processing for SEM. Processing soft tissues for scanning electron microscopy (SEM) presents many problems depending on the size, shape and type of specimen. Cohen (1974) presented an overall view of this problem. Methods for handling cells in suspension have been suggested by Baker & Princen (1975), Newell & Roath (1975) and Rostgaard & Christensen (1975). Tissue culture specimens are also difficult to process for SEM. These may be long-term cultures such as fibroblasts or kidney cells, or short-term preparations such as the collecting of white blood cells or bacteria on coverslips. Nemanic (1972) described a method using Tygon tubing with slits to hold the coverslips after incubation. Perecko et al. (1973) gave details of a holder for processing six small (6 mm) coverslips after incubation. Rice et al. (1976) designed several complex multipurpose carriers for 9 × 22 mm coverslips and for cells on silver or cellulose membrane filters. Broers et al. (1975) used a mesh receptacle for bacteria attached to silicon dioxide slivers. Many investigators simply process pieces of plastic cut from the tissue culture dish itself. Using the plastic directly avoids the problems of incubating cells on glass coverslips. Coverslips are fragile and difficult to remove from the culture dish or flask. Each coverslip must be processed individually, increasing the risk of breakage and limiting the number which can be done at one time. For processing larger tissues there are commercial baskets available. Cohen (1974) illustrated mesh baskets for odd sizes and shapes of soft tissues. We have devised a simple holder which facilitates the use of 12 mm 0 grade coverslips in several types of culture dishes. We have developed a multipurpose carrier which increases the number of coverslips which can be processed for SEM and reduces the number of manipulations needed for each individual coverslip. This carrier can be used for histological and histochemical studies as well as SEM processing. We have modified Cohen's technique (1974) for mesh baskets for larger soft tissue specimens.     

Micromorphological characterization of zinc/silver particle composite coatings

The aim of this study was to evaluate the three‐dimensional (3D) surface micromorphology of zinc/silver particles (Zn/AgPs) composite coatings with antibacterial activity prepared using an electrodeposition technique. These 3D nanostructures were investigated over square areas of 5 μm × 5 μm by atomic force microscopy (AFM), fractal, and wavelet analysis. The fractal analysis of 3D surface roughness revealed that (Zn/AgPs) composite coatings have fractal geometry. Triangulation method, based on the linear interpolation type, applied for AFM data was employed in order to characterise the surfaces topographically (in amplitude, spatial distribution and pattern of surface characteristics). The surface fractal dimension D_f, as well as height values distribution have been determined for the 3D nanostructure surfaces. Microsc. Res. Tech. 78:1082–1089, 2015. © 2015 The Authors published by Wiley Periodicals, Inc.     

MRT letter: Localization of endogenous hydrogen peroxide by modified processes of sample preparation for transmissionelectron microscope in Escherichia coli

The bacterial endogenous hydrogen peroxide (H₂O₂) was detected cytochemically by its reaction with cerium chloride (CeCl₃) to produce electron‐dense deposits of cerium perhydroxides. The sequence of fixation and CeCl₃ staining of H₂O₂ in the processing of transmission electron microscope (TEM) sample preparation is crucial to the localization of endogenous H₂O₂ in Escherichia coli. In this study, results confirmed that the process that fixation simultaneously with CeCl₃ staining provided optimum effects for H₂O₂ localization in E. coli. The modified process of TEM provides very efficient protection for H₂O₂ localization and more accurate quantization for the H₂O₂ accumulation in bacterial cells. Microsc. Res. Tech., 2013. © 2012 Wiley Periodicals, Inc.     

Myelin phagocytosis by macrophages and nonmacrophages during Wallerian degeneration

The literature concerning Schwann cells (SCs) and macrophages in myelin phagocytosis during Wallerian degeneration is reviewed. SCs carry out the first step in the removal of myelin by segmenting myelin and then incorporating the degraded myelin. The recruited macrophages then join in the myelin-phagocytosis event, appearing to make full use of their original phagocyte abilities until the end of myelin clearance. The molecular mechanisms of the two cells underlying myelin phagocytosis are thought to be different; myelin phagocytosis by SCs being lectin-mediated, i.e., opsonin-independent, whereas that of macrophages is mainly opsonin-dependent. It is important to note that SCs and macrophages cooperatively accomplish myelin phagocytosis.     

The use of silver nitrate as a stain for scanning electron microscopy of arterial intima and paraffin sections of kidney

The suitability of silver nitrate as a stain for scanning electron microscopy was investigated. Accordingly en face preparations of arterial intima were impregnated with silver nitrate, fixed with formalin, and coated with platinum-palladium alloy. In SEM images, the silver lines surrounding the endothelial cells, and the deposits on the intima appear as white lines or dots on a darker background. Similar results were noted for nitrocellulose-embedded endothelium. Paraffin sections of kidney treated with silver nitrate (von Kossa's stain) were also examined. Deposits of the calcium salts of phosphates and carbonates (stained with silver nitrate) were easily differentiated from other tissue components. Results were compared with those obtained with the light microscope. The scanning electron microscopic examination provided a finer definition of the silver granules relative to the surrounding architecture. The limitations and advantages of these techniques and possible further applications of silver nitrate as a stain for scanning electron microscopy are discussed. The use of silver nitrate as a stain for some SEM preparations is recommended.     

Stem cells in the development and differentiation of the human adrenal glands

There are no studies on stem cells (SCs) and development and differentiation (DD) of the human adrenal glands. The SCs in DD of the adrenal glands were herein investigated histochemically and immunohistochemically in 18 human embryonic adrenal glands at gestational week (GW) 7–40. At 7 GW, the adrenal glands were present, and at 7 GW, numerous embryonic SCs (ESCs) are seen to create the adrenal cortex. The ESCs were composed exclusively of small cells with hyperchromatic nuclei without nucleoli. The ESCs were positive for neural cell adhesion molecule, KIT, neuron‐specific enolase, platelet‐derived growth factor receptor‐α, synaptophysin, and MET. They were negative for other SC antigens, including chromogranin, ErbB2, and bcl‐2. They were also negative for lineage antigens, including cytokeratin (CK)7, CK8, CK18, and CK19, carcinoembryonic antigen, carbohydrate antigen 19‐9, epithelial membrane antigen, HepPar1, mucin core apoprotein (MUC)1, MUC2, MUC5AC, and MUC6, and cluster differentiation (CD)3, CD45, CD20, CD34, and CD31. The Ki‐67 labeling index (LI) was high (Ki‐67 LI = around 20%). α‐Fetoprotein was positive in the ESCs and adrenal cells. The ESC was first seen in the periphery of the adrenal cortex at 7–10 GW. The ESC migrates into the inner part of the adrenal cortex. Huge islands of ESC were present near the adrenal, and they appeared to provide the ESC of the adrenal. At 16 GW, adrenal medulla appeared, and the adrenal ESCs were present in the periphery or the cortex, in the cortical parenchyma, corticomedullary junctions, and in the medulla. The adrenal essential architecture was established around 20 GW; however, there were still ESCs. At term, there are a few ESCs. These data suggest that the adrenal glands were created by ESCs. Microsc. Res. Tech., 78:59–64, 2015. © 2014 Wiley Periodicals, Inc.