Ferrocyanide-reduced Osmium Research Articles

Presence of Surfactant Lamellar Bodies in Normal and Diseased Sinus Mucosa

Background:Pulmonary surfactant originates from phospholipid lamellar bodies secreted from the type II epithelial cell of the alveolus. In the lower airway, surfactant optimizes surface tension and oxygen exchange, decreases mucus viscosity and aids in mechanical elimination of inhaled pathogens. In addition to the lung, lamellar bodies have been identified in many other cell types throughout the human body. However, no prior studies have identified lamellar bodies in human sinus mucosa. Objectives: We performed ultrastructural studies to assess whether lamellar bodies are present in the human sinus in a variety of diseased and normal epithelium. Methods:We biopsied sinus mucosa from 5 subjects, 1 each with allergic fungal sinusitis, eosinophilic mucin rhinosinusitis, cystic fibrosis, frontal sinus mucocele, and cerebrospinal fluid leak (healthy control). Mouse lung served as a positive control. Specimens were prepared using ferrocyanide-reduced osmium tetroxide and thiocarbohydrazide for fixation (R-OTO method) to avoid extraction of phospholipids during dehydration and were viewed with transmission electron microscopy. Results:We identified lamellar bodies in the sinus mucosa of all patients. Additionally, preservation of mouse lung lamellar bodies confirms that the R-OTO method is a valid technique to preserve these structures. Conclusions:We describe a simpler, faster technique for identification of cellular phospholipid components than those used previously. Definitive identification of these lamellar bodies within ciliated pseudostratified epithelium of the upper airway indicates that surfactant may have a role in sinus function and pathophysiology.

Thin Is Better!: Ultrathin Cryosection Immunocytochemistry

In immunofluorescence microscopy (IFM), the repression of out of focus fluorescence signal is crucial in order to obtain high-resolution images. One option to acquire high vertical resolution (z-axis resolution) is to produce optical sections with a confocal microscope. The z-axis resolution usually obtained with confocal microscopy of biological samples is about 500 nm. Another option is to produce very thin sections with a cryo-ultramicrotome (physical sections). The ultrathin cryosections we employ are about 100 nm in thickness: thus all of the fluorescence must come from within this 100 nm thickness. The use of ultrathin cryosections permits the acquisition of extremely high-quality images and minimizes the possibility for false localization in IFM (Fig. 1). Ultrathin cryosections can be applied to immunoelectron microscopy (IEM) as well as IFM (Fig. 2). We show new methods of ultrathin cryosection immunocytochemistry(1-3). Human full-term placentas were fixed with 4% paraformaldehyde, solidified with 10% gelatin, infiltrated with 2.3 M sucrose, and then frozen in liquid nitrogen. Ultrathin cryosections were cut with a cryo-ultramicrotome and then transferred to glass cover slips for IFM or to nickel grids for IEM. Cryosections were incubated with mouse anti-p230, a trans-Golgi network marker, and subsequently incubated with Alexa 488-labeled goat anti-mouse IgG or with goat anti-mouse 5-nm colloidal gold particles. For visualization and preservation of ultrastructure of cryosections at the electron microscopic level, the sections on grids were postfixed with ferrocyanide-reduced osmium and then stained with uranyl acetate and lead citrate in polyvinyl alcohol(1). Ultrathin cryosection immunocytochemistry should be an important technique for functional genomics research, especially for the analysis of the in situ expression of target molecules(2,3).

Piecemeal degranulation (PMD) morphology in feline circulating eosinophils

Buffy coat preparation from six cats with 600–4560 circulating eosinophils/μL was collected by either blood centrifugation or sedimentation, fixed in 2.5% glutaraldehyde, post-fixed in either 1% osmium or in 1.5% potassium ferrocyanide-reduced osmium, ultra-sectioned and examined by transmission electron microscopy. Ultrastructural changes of piecemeal degranulation (PMD), which is a mechanism of eosinophil granule contents release indicative of eosinophil activation, were observed in specific granules from all the samples examined. The spectrum of PMD included coarsening of the granule matrix, budding vesicles, fragmented granule cores and lucent granules. The number of presumably activated eosinophils with ultrastructural evidence of PMD did not correlate with the level of eosinophilia. The lack of correlation suggested that, analogously with humans, blood eosinophil count might not represent the best criterion to evaluate the contribution of eosinophils to tissue damage in certain feline eosinophil-associated diseases.

Ultrastructural and ultracytochemical features of secretory granules in the ampullary epithelium of the hamster oviduct.

The epithelium of mammalian oviducts consists mainly of ciliated and non-ciliated secretory cells. In some mammals, secretory products originating from oviductal secretory cells have been shown to bind to the surface of, or accumulate within, ovulated eggs and/or developing embryos. These findings suggest that the secretions of the oviductal epithelial cells may play an important role in reproductive and developmental events that occur in the oviduct. In the present study, ultrastructural and cytochemical features of secretory cells in the hamster ampullary epithelium were shown by routine electron microscopy, lectin-gold cytochemistry and both conventional freeze-fracture and rapid-freezing techniques with special reference to the organizational aspects of their secretory granules. The use of ferrocyanide-reduced osmium tetroxide as a post-fixative in the Epon embedment of ampullary tissue samples also proved to be advantageous especially in revealing the carbohydrate contents of certain cellular compartments. The most conspicuous characteristic of the secretory cells, based on their staining property, was the presence of two types of secretory granules: those with a homogeneous electron-dense matrix and those with an electron-lucent matrix. Under favorable conditions, distinct features of the organizational arrangement of a crystalline lattice inside the secretory granules were also revealed. This well organized crystalline lattice shown in sections of Epon-embedded oviductal tissue was confirmed by examination of replicas of freeze-fractured oviducts prepared by the rapid-freezing technique. We also demonstrated with high resolution lectin-gold cytochemistry the intracellular distribution of lectin-binding glycoconjugates in the secretory cells of the hamster oviductal ampulla often in a linear array following the crystalline lattice. The results obtained in this study, taken together, provide insight into a possible link of the internal topographical features of oviductal secretory granules along with the cytochemical properties of their contents to the anticipated regulatory mechanism underlying their process of secretions.

Cytochemical study of glycogen in myoblasts of the chick embryo myotome

Potassium ferrocyanide-reduced osmium has been used as secondary fixative to study the presence of glycogen in different tissues. However, this method has not been previously applied to skeletal muscle development research. This study reports the use of this method to analyze the structure and cytoplasmic distribution of glycogen particles in the myoblasts of the brachial myotome of chicken embryos at 51-105 h. of incubation (Hamburger and Hamilton stages 16 to 25).In those sections post-fixed with potassium-ferrocyanide reduced osmium it is observed that glycogen particles stain in contrast to the rest of the structures of the cell (Figs. 1,2). In those sections processed for T.E.M., following conventional methods, glycogen particles are occasionally observed in less contrast and number that those with potassium-ferrocyanide reduced osmium. An explanation for this differences might be in the effects of potassium-ferrocyanide reduced osmium fixation preventing the loss of glycogen during dehydration and staining with uranyl acetate. We conclude that this cytochemical method may be specific for glycogen detection.

Ultrastructural study of proliferating cells with an improved immunocytochemical detection of DNA-incorporated bromodeoxyuridine.

We designed an improved method to observe proliferating cells with well-preserved ultrastructure. After IP injection of bromodeoxyuridine (BrdU) into rats, the pituitaries were fixed in 4% paraformaldehyde with 0.05% or 0.2% glutaraldehyde and post-fixed with ferrocyanide-reduced osmium. They were embedded in LR White and polymerized by heat. BrdU incorporated into DNA was detected with a commercial anti-BrdU monoclonal antibody (MAb) by the immunogold or the immunogold-silver staining method. Using these methods, proliferating cells labeled by BrdU were observed with well-preserved ultrastructure. By light microscopy, the number of labeled cells was almost the same regardless of the fixative used. By electron microscopy, localization of gold particles that indicate incorporated BrdU varied according to the cells and was mainly observed in two patterns, one in which gold particles were localized in condensed chromatin scattered in the nucleus and the other in which gold particles were dispersed evenly all over the nucleus. These results showed that with our improved method fine ultrastructure and good immunoreactivity of BrdU can be obtained in proliferating cells. We consider that this method is very useful for ultrastructural study of cell proliferation and differentiation.

Kurloff cell ultrastructure after combined formaldehyde-cetylpyridinium chloride fixation and high-iron diamine staining

This study deals with the ultrastructure of the chondroitin sulphate proteoglycans of the Kurloff body, a large lysosomal organelle that stains metachromatically with Toluidine Blue and which is present in Kurloff cells (a blood cell unique to the guinea pig). Splenic tissues were fixed with 1% cetylpyridinium chloride (CPC) added to 4% paraformaldehyde and examined either after Spicer's high-iron diamine staining for sulphated anionic sites followed by post-fixation with ferrocyanide-osmium tetroxide or after a simple post-fixation with ferrocyanide-osmium tetroxide. CPC-precipitated sulphated sites were preferentially located at the periphery of the Kurloff body but, unexpectedly, were absent in the central matrix. Although their electron opacity was lower, these anionic sites were readily observable in the absence of HID-staining after sole post-fixation by ferrocyanide-reduced osmium. CPC-precipitated sulphated anionic sites were either associated with the myelin figures or constituted unexpected structures. They contained (i) tightly-stacked lamellae, with a very regular 4 nm periodicity, and (ii) groups of 2, 3, 4 short dense lines with a 3-5 nm periodicity. By taking into account the susceptibility of these HID-reactive structures towards chondroitinase ABC, these different sulphated components were assumed to be related to the proteochondroitin-4-sulphate previously characterized as the only major sulphated glycoconjugate of the Kurloff cell. Their colocalization with phospholipidic structures was suggested following observation of sections treated by a chloroform-methanol mixture.

The use of osmicated tissues for Lowicryl K4M embedding.

Tissue chopper slices of rat incisor, rat parotid gland, and chicken tibiae, fixed with 1% glutaraldehyde, were post-fixed with potassium ferrocyanide-reduced osmium tetroxide, dehydrated with methanol, and conventionally embedded in Lowicryl K4M at -20 degrees C. The tissues showed an ultrastructural appearance comparable with that of their Epon-embedded counterparts and, in particular, the Golgi apparatus was well defined. Furthermore, Lowicryl K4M-embedded osmicated tissues permitted both post-embedding lectin-gold cytochemistry and immunogold labeling.

Ultrastructure of the principal and accessory submandibular glands of the common vampire bat

The principal and accessory submandibular glands of the common vampire bat, Desmodus rotundus, were examined by electron microscopy. The secretory endpieces of the principal gland consist of serous tubules capped at their blind ends by mucous acini. The substructure of the mucous droplets and of the serous granules varies according to the mode of specimen preparation. With ferrocyanide-reduced osmium postfixation, the mucous droplets are moderately dense and homogeneous; the serous granules often have a polygonal outline and their matrix shows clefts in which bundles of wavy filaments may be present. With conventional osmium postfixation, the mucous droplets have a finely fibrillogranular matrix; the serous granules are homogeneously dense. Mucous cells additionally contain many small, dense granules that may be small peroxisomes, as well as aggregates of 10-nm cytofilaments. Intercalated duct cells are relatively unspecialized. Striated ducts are characterized by highly folded basal membranes and vertically oriented mitochondria. Luminal surfaces of all of the secretory and duct cells have numerous microvilli, culminating in a brush borderlike affair in the striated ducts. The accessory gland has secretory endpieces consisting of mucous acini with small mucous demilunes. The acinar mucous droplets contain a large dense region; the lucent portion has punctate densities. Demilune mucous droplets lack a dense region and consist of a light matrix in which fine fibrillogranular material is suspended. A ring of junctional cells, identifiable by their complex secretory granules, separates the mucous acini from the intercalated ducts. The intercalated ducts lack specialized structure. Striated ducts resemble their counterparts in the principal gland. As in the principal gland, all luminal surfaces are covered by an array of microvilli. At least some of the features of the principal and accessory submandibular glands of the vampire bat may be structural adaptations to the exigencies posed by the exclusively sanguivorous diet of these animals and its attendant extremely high intake of sodium chloride.

Brush cells of the mouse gallbladder

The brush cells (BC) of the mouse gallbladder were studied using light and electron microscopy (transmission and scanning) to determine their shape and distribution. Specimens were fixed in glutaraldehyde and postfixed in ferrocyanide-reduced osmium tetroxide. BC selectively stained with toluidine blue could be identified by means of light microscopy and subsequently studied in serial semithin and ultrathin sections. The results revealed that the shape of the BC is flask-like. A slender, occasionally branched cytoplasmic process emerges from the bulk cell body and extends through the basal region of neighboring epithelial elements to the basement membrane. Examination of the entire gallbladder epithelial surface by scanning electron microscopy revealed that the BC are numerous in the neck region of the organ but only scanty or even absent in wide areas of the corpus region. Their number increases again in the fundic region. These results demonstrate a preferential regional distribution of BC in the gallbladder, which is discussed in relation to a possible functional significance of the BC.

Use of lead aspartate block staining in quantitative EM autoradiography of phospholipids: application to myelinating peripheral nerve.

For quantitation of electron microscope (EM) autoradiographs, micrographs must contain clear images which are relatively free of heavy metal precipitates. Satisfactory contrast is usually obtained by staining individual ultra-thin sections with lead citrate. It was recently reported that sequential block staining of tissue with ferrocyanide-reduced osmium tetroxide and lead aspartate produced excellent contrast for EM autoradiography, with sections relatively free of lead precipitate. This protocol avoids the manipulation involved in staining individual ultra-thin sections. We have adapted this method to quantitative EM autoradiographic studies, primarily of phospholipid metabolism in peripheral nerve. We show that block staining with lead aspartate provides: (a) ultrastructural contrast of routinely high quality for myelinated peripheral nerve; (b) high (greater than 98%) retention of glycero-labeled lipid during dehydration and embedment; and (c) a distribution of de novo tritiated glycerol-labeled lipid in ultra-thin sections that is quantitatively identical to the distribution recorded for samples stained by the more laborious post-embedment method. During a 2-hr labeling period in vivo, tritiated glycerol is incorporated into phosphatidylcholine (44%), phosphatidylethanolamine (22%), other phospholipids (16%), and neutral lipids (15%). The analysis of grain distribution in developing sciatic nerve labeled for 2 hr with tritiated glycerol demonstrates that myelinating Schwann cells play the major role in synthesis of endoneurial lipids. Lipid synthesis in myelinated fibers is localized in perinuclear regions of Schwann cell cytoplasm. These regions lie external to compact myelin. Unmyelinated fibers and other endoneurial cells independently incorporate glycerol into lipids.

Application of the ferrocyanide-reduced osmium method for mineralizing cartilage: Further evidence for the enhancement of intracellular glycogen and visualization of matrix components

The ferrocyanide-reduced osmium (FRO) fixation method was applied to neonatal mouse mandibular condylar cartilage for its processing for electron microscopy. The results were compared to those obtained by the conventional glutaraldehyde-osmium tetroxide fixation method. Three different stages in the life cycle of condylar cartilage cells were examined. FRO enabled the visualization of delicate fibrillar mesh in the matrix of all three zones of the cartilage, resulting in a dense appearance of the intercellular matrix. The classical stellate shape of matric granules seen in cartilage fixed with glutaraldehyde-osmium tetroxide was not observed in FRO-processed tissues. Chondrocytes that were FRO-processed almost entirely filled their lacunar space. In their pericellular area, fibrillar material and electron-dense aggregates could be demonstrated by the FRO method. As a conclusion of this study, it is recommended to supplement a conventional protocol with the FRO fixation method for routine and research purposes.

Schistosoma mansoni: Ultrastructural demonstration of a miracidial glycocalyx that cross-reacts with antibodies raised against the cercarial glycocalyx

Cercariae are covered by a glycocalyx that is highly antigenic. Here, we have examined the surface of miracidia for a similar structure. The miracidia are covered by epithelial plates and syncytial ridges. By transmission electron microscopy, the plates and ridges were covered by a 0.5-μm-thick glycocalyx composed of a mesh of 9- to 10-nm fibrils that were stained by ruthenium red delivered in the aldehydes or ferrocyanide-reduced osmium tetroxide. Rabbit antibodies prepared against phenol extracted and chromatographed cercarial glycocalyx were detected by immunoelectron microscopy with secondary antibodies conjugated to horseradish peroxidase. Reaction product bound to both the miracidial and cercarial glycocalyx. In addition, the outer leaflets of the cercarial tegumental membrane and membranes of the miracidial surface structures, including plates, ridges, terebratorium, and sensory papillae, had reaction product. Controls incubated with nonspecific rabbit serum had no reaction product. By indirect immunofluorescence, antibodies against the cercarial glycocalyx stained both plates and ridges. As the miracidia transformed to sporocysts, the glycocalyx remained associated with the plates as they were sloughed. These studies demonstrate that miracidia possess a glycocalyx similar in structure and antigenicity to the cercarial glycocalyx.

Periodic acid Schiff--p phenylenediamine staining of glycogen in chondrocytes. A new combination which improves both cellular detail and glycogen identification.

This study reports a method whereby glycogen is identified in the chondrocytes of the secondary center of ossification prior to mineralization. The use of new fuchsin rather than basic fuchsin on one micron Spurr sections of femoral head cartilage fixed with potassium ferrocyanide-reduced osmium produced excellent identification of glycogen and when followed by p phenylenediamine, intensified cellular detail.

Influence of cetylpyridinium chloride on the ultrastructural appearance of sulphated glycosaminoglycans in human colonic mucosa.

The effect of adding cetylpyridinium chloride to the fixative on the preservation of sulphated glycosaminoglycans (SGs) was studied in human normal colonic mucosa. SGs were visualized at the ultrastructural level through the application of Spicer's High Iron Diamine (HID) technique followed by a post-fixation with potassium ferrocyanide-reduced osmium tetroxide. SGs were mainly localized in basement membranes of epithelium and capillary wall and along collagen fibers. The morphology of the reactive sites depended on the presence of cetylpyridinium chloride, SGs being granular in absence of the salt and more or less elongated when cetylpyridinium chloride was added to the fixative. We suggest that the use of cetylpyridinium chloride during fixation may help to preserve SG molecule at the ultrastructural level.

Ultrastructure of the submandibular gland in the rabbit.

The secretory endpieces of the rabbit submandibular gland are unusual in that they consist of seromucous acini (not demilunes) that empty into serous tubules that in turn drain into intercalated ducts. Seromucous granules consist of a moderately dense spherule in a fibrillogranular matrix. Serous granules contain a feltwork of filaments, which are liberated as a tangled skein during exocytosis. Peculiar granulated cells that have secretory granules of complex morphology are present at each end of the serous tubules. Intercalated ducts are, cytologically speaking, relatively simple, but the duct cells may contain a few oblong secretory granules. Striated ducts are typical in structure, although postfixation with ferrocyanide-reduced osmium reveals significant amounts of glycogen in the basal processes. Modified mitochondria are present in striated duct cells, but their frequency varies from rabbit to rabbit. Such mitochondria contain either an array of parallel, rigid cristae linked by intermembranous bridges, or a bundle of helical filaments within an expanded crista. Interspersed with the striated duct cells, especially near the duct origin, are some highly vacuolated cells with sparse mitochondria. Excretory ducts consisting of stratified columnar (sometimes pseudostratified) epithelium often show bleb formation of the luminal surface of the tall cells.

The sarcoplasmic reticulum of mouse heart: Its divisions, configurations, and distribution

The sarcoplasmic reticulum (SR) is a prominent, highly ramified component of mouse myocardial cells. The use of ferrocyanide-reduced osmium tetroxide (OsFeCN) as a postfixative solution facilitates appreciation of both its extent and three-dimensional architecture. We have found that the individual volume fractions (Vv) of myofibrils, mitochondria, and SR are similar in cells of the right and left ventricular walls.Vv(total SR) is approximately 7%, a value considerably larger than previously reported. We attribute this disparity in large part to the recognition factor which comes into play with OsFeCN-treated tissue. Previous observations pertaining to the stereology of myocardial SR have likely substantially underestimated both volume fraction and surface density of this membrane system, since none to this point has utilized specific staining such as that conferred by the OsFeCN regimen. Our stereological measurements of different depths of the ventricular cell indicate that although considerable differences are found between SR configuration at peripheral and deep cell levels, no significant difference exists between the volume fractions of either the total SR or its individual constituents. Two different stereologic regimens gave close agreement on volume fractions of the various SR segments; the majority (approximately 92%) of the total SR is network SR, whereas the remainder is composed of the various categories of junctional SR (peripheral, apposed to the surface sarcolemma;interior, complexed with the transverse-axial tubular system;corbular, existing free of sarcolemmal contact). In the adult mouse, interior junctional SR greatly preponderates the other types of junctional SR; corbular SR is qualitively assessed to be a far more common component of atrial cells than of ventricular cardiomyocytes.

Tridimensional architecture of the Golgi apparatus in the atrial muscle cell of the rat.

The tridimensional structure of the Golgi apparatus of atrial muscle cells has been studied in thin and thick sections with low- and high-voltage electron microscopes. Cardiac tissue was inpregnated with osmium, stained to demonstrate phosphate activity (i.e., nicotinamide adenine dinucleotide phosphatase or NADPase; thiamine pyrophosphatase or TPPase; cytidine monophosphatase or CMPase) or postfixed and stained with potassium ferrocyanide-reduced osmium. At low power, in thick (3-10 micron) sections, the cisosmiophilic element and the NADPase- and the TPPase-positive saccules each appeared as a continuous irregular ribbon that formed, at the two poles of the nucleus, two conical masses connected to each other by beltlike bands encircling the nucleus. At higher magnifications, the continuous Golgi apparatus showed saccular regions along its length connected by short intersaccular tubular regions. In the saccular regions, the following five superimposed elements formed a stack: (1) the cis-osmiophilic network of anastomosed tubules; (2) a chromophobic, dilated saccule perforated with numerous pores; (3) a thin NADPase-positive saccule showing few pores; (4) a thin TPPase-positive saccule perforated with numerous minute pores; and (5) a CMPase-positive transelement that showed saccular and tubular regions and was often partly separated from the overlying saccule. In the intersaccular tubular regions, membranous tubules connected and bridged saccules of two adjacent saccular regions. Secretory granules usually appeared in this region as dilations of the tubules connected to all elements of the Golgi stack except the cis-osmiophilic element.

The use of osmium-thiocarbohydrazide-osmium (OTO) and ferrocyanide-reduced osmium methods to enhance membrane contrast and preservation in cultured cells.

The preservation and contrast of membranous structures in cultured cells using various postfixation procedures prior to embedding have been investigated. These include routine OsO4, ferrocyanide-reduced OsO4, osmium-thiocarbohydrazide-osmium (OTO), and ferrocyanide-reduced osmium-thiocarbohydrazide-ferrocyanide-reduced osmium (R-OTO). With standard ethanol-Epon dehydration/embedding techniques, a dramatic improvement in both membrane contrast and preservation of bilayer membrane structure was achieved using preembedding OTO in cultured cells. R-OTO yielded similar enhanced preservation and contrast of membranes. Both of these methods also resulted in an increase in the contrast of diaminobenzidine reaction product from horseradish peroxidase activity, and of lipid droplets and lipoprotein particles. However, R-OTO did not cause the same increase in the density of proteinaceous elements as was seen with the OTO method. Ferrocyanide-reduced osmium alone showed significant advantages for quantitation of immunocytochemistry using ferritin labels with bismuth subnitrate counterstain. These methods should have general usefulness for the preservation of lipid-containing structures in cultured cells.

Development of Intracellular Membrane Systems in the Columnar Epithelium of the Urinary Bladder of the Euryhaline Goby Gillichthys mirabilis: (urinary bladder/intracellular membranes/euryhaline teleost/prolactin/osmoregulation).

The membrane systems in "columnar cells" of the goby urinary bladder were studied after staining with ferrocyanide-reduced osmium tetroxide (Karnovsky). In addition to the endoplasmic reticulum, two distinct systems of membranes were observed: 1) a vesiculotubular system made up of vesicles and short tubules located between the Golgi area and the apical membrane and 2) well-developed infoldings of the laterobasal plasma membrane which form either complete or fenestrated sheets. Adaptation to 5% seawater or prolactin exposure of seawater fish induces a proliferation of these membrane systems and, in particular, of the complete infoldings of the laterobasal plasma membrane. These observations suggest high activity of these bladder cells in osmoregulatory adjustments to hypotonic environments. The divergence between cytological and physiological indicators of activity is considered.