OsO4 Postfixation Research Articles

The effects of osmium tetroxide post-fixation and drying steps on leafy liverwort ultrastructure study by scanning electron microscopy.

Leafy liverwort is one of the most abundant and diverse plants in Indonesia. Their high variation and beneficial secondary metabolites contained in the oil bodies have attracted researchers' attention. The ultrastructural analysis of leafy liverworts is important as a means of species identification and also for further exploration of their oil bodies. However, the optimization of the preparation steps for observing leafy liverworts by SEM is necessary to avoid sample destruction. Fixation and drying play important roles in maintaining a sample's structure as close to its natural state as possible. Thus, in this study, we evaluated the effect of 4% Osmium tetroxide (OsO4 ) and drying on leafy liverworts ultrastructure. Microlejeunea, Acrolejeunea, and Frullania were fixed with 2.5% glutaraldehyde. Some samples were then post-fixed with 4% OsO4 , while the rest were directly dehydrated with an ethanol series and then subjected to different drying methods, i.e. air drying, freeze drying, and drying with hexamethyldisilazane (HMDS). According to the data obtained, post-fixation with 4% OsO4 could better maintain the integrity of the samples and enhance the contrast of leafy liverwort SEM images. In addition, samples dried with HMDS showed more detailed structures compared to those that were air dried. Different ultrastructure were found among the different leafy liverworts observed by SEM. Our data suggested the advantages of SEM in providing ultrastructure information on leafy liverworts as well as the optimum conditions to observe them with less deformation. OsO4 post-fixation could enhance the contrast of leafy liverwort SEM images and maintain the structure of the samples. Drying with HMDS provided a convenient way for rapid SEM preparation with less structural distortion.

Quantitating morphological changes in biological samples during scanning electron microscopy sample preparation with correlative super-resolution microscopy.

Sample preparation is critical to biological electron microscopy (EM), and there have been continuous efforts on optimizing the procedures to best preserve structures of interest in the sample. However, a quantitative characterization of the morphological changes associated with each step in EM sample preparation is currently lacking. Using correlative EM and superresolution microscopy (SRM), we have examined the effects of different drying methods as well as osmium tetroxide (OsO4) post-fixation on cell morphology during scanning electron microscopy (SEM) sample preparation. Here, SRM images of the sample acquired under hydrated conditions were used as a baseline for evaluating morphological changes as the sample went through SEM sample processing. We found that both chemical drying and critical point drying lead to a mild cellular boundary retraction of ~60 nm. Post-fixation by OsO4 causes at least 40 nm additional boundary retraction. We also found that coating coverslips with adhesion molecules such as fibronectin prior to cell plating helps reduce cell distortion from OsO4 post-fixation. These quantitative measurements offer useful information for identifying causes of cell distortions in SEM sample preparation and improving current procedures.

FE-SEM and VP-SEM imaging of human calcified aortic valves: conventional vs Ionic Liquid innovative techniques

Conventional FE-SEM protocol for calcified aortic valves (CAVs) consist of following steps: glutaraldehyde fixation, OsO4 post-fixation, dehydration in alcohol series, critical point drying and finally sputter coating. CAVs can be observed in their native state (fixed in glutaraldehyde with and without post-fixation in OsO4) by Variable Pressure-SEM (range 6- 650 Pa). Gas presence allows an inferior resolution (low signal to noise ratio), however there is the possibility to perform EDS elemental analysis without background noise due to sputter coating. Recently Ionic liquids (IL, salts in the liquid state at room temperature) were used as suppliers of electronic conductivity with insulating properties, so we have tested their ability to replace sputter coating on CAVs in high vacuum condition. Samples fixed in glutharaldehyde 2,5% in PBS with and without OsO4 post-fixation treated with ionic liquid (Hitachi HILEM® IL 1000) were compared with samples treated with conventional FE-SEM procedures. Several IL concentration (range from 5% to 20%) were tested, different operating voltages (range from 3 to 20Kv) were used. This novel technology requires a high degree of customization for each sample type. In our opinion fixation in glutaraldehyde with OsO4 post-fixation is recommended to preserve finest details, moreover residual liquid elimination is important to increase resolution and avoid beam interference as linear markings. Setting of a proper accelerating voltage allows to correctly visualize the surface topography. Processing CAVs with IL with respect to conventional FE-SEM is useful for several reasons. Mainly this method is time saving (and cost saving), secondary the same sample can be processed for transmission electron microscopy after SEM observations (allowing correlative microscopy), finally EDS can be performed without background noise due to sputter coating. Perhaps now this technique can not completely replaces the conventional SEM in terms of resolution but in our opinion rapid technical improvement can further reduce this gap.

Ultrastructure of the elementary fibril in extended and condensed chromatin and in metaphase chromosomes

The chromatin Ultrastructure of nuclei in ultrathin sections of root meristem in <i>Cucurbita pepo</i> - the species poor in DNA, and in <i>Haemanthus katharinae</i> - rich in DNA was compared. Four combinations of fixation and embedding were used: 1) OsO<sub>4</sub> (pH 7.2), methacrylates; 2) OsO<sub>4</sub> (pH 7.2), Epon; 3) OsO<sub>4</sub> (pH 6.8), Epon; 4) glutaraldehyde (pH 7.2-7.4) with OsO4 postfixation, Epon embedding. Most suitable was OsO<sub>4</sub> fixation (pH 7.2) and embedding in Epon because of the smallest dispersion of dimensions of chromatin fibrils and good visibility of their substructure. Fibrils of condensed chromatin, components of chromocenters (heterochromatin) in <i>Cucurbita pepo</i>, bands of chromonemata visible in the light microscope and metaphase chromosomes in <i>Haemanthus katharinae</i> showed a diameter of about 11.5 nm after OsO<sub>4</sub> fixation, or 14-19 nm after glutaraldehyde. Fibrils of extended chromatin (euchromatin) in <i>Cucurbita pepo</i> are thinner, their diameter is about 10 nm after OsO<sub>4</sub> fixation and Epon embedding, and about 14.5 nm after glutaraldehyde. There are no visible differences in the width of elementary fibrils after OsO<sub>4</sub> fixation and embedding in methacrylates. In both species the fibrils of extended chromatin are built up of one coiled deoxyribonucleohistone (DNH) thread about 2.3 nm in diameter, while the fibrils of condensed chromatin consist of two DNH threads, each about 3 nm in diameter. The diameter of DNH threads is determined neither by the fixative nor by the way of embedding; they are less visible in the material embedded in methacrylates. The results obtained show that the structure of chromatin fibrils depends on the kind of chromatin, but not on DNA content. In <i>Haemanthus katharinae</i>, the species rich in DNA, the greater amount of chromatin appears in condensed form.

Ultrastructure of skin from Refsum disease with emphasis on epidermal lamellar bodies and stratum corneum barrier lipid organization.

Classic Refsum disease (RD) is a rare, autosomal recessively-inherited disorder of peroxisome metabolism due to a defect in the initial step in the alpha oxidation of phytanic acid (PA), a C16 saturated fatty acid with four methyl side groups, which accumulates in plasma and lipid enriched tissues (please see van den Brink and Wanders, Cell Mol Life Sci 63:1752-1765, 2006). It has been proposed that the disease complex in RD is in part due to the high affinity of phytanic acid for retinoid X receptors and peroxisome proliferator-activated receptors. Structurally, epidermal hyperplasia, increased numbers of cornified cell layers, presence of cells with lipid droplets in stratum basale and reduction of granular layer to a single layer have been reported by Blanchet-Bardon et al. (The ichthyoses, SP Medical & Scientific Books, New York, pp 65-69, 1978). However, lamellar body (LB) density and secretion were reportedly normal. We recently examined biopsies from four unrelated patients, using both OsO4 and RuO4 post-fixation to evaluate the barrier lipid structural organization. Although lamellar body density appeared normal, individual organelles often had distorted shape, or had non-lamellar domains interspersed with lamellar structures. Some of the organelles seemed to lack lamellar contents altogether, showing instead uniformly electron-dense contents. In addition, we also observed mitochondrial abnormalities in the nucleated epidermis. Stratum granulosum-stratum corneum junctions also showed co-existence of non-lamellar and lamellar domains, indicative of lipid phase separation. Also, partial detachment or complete absence of corneocyte lipid envelopes (CLE) was seen in the stratum corneum of all RD patients. In conclusion, abnormal LB contents, resulting in defective lamellar bilayers, as well as reduced CLEs, likely lead to impaired barrier function in RD.

Direct evidence of lipid translocation between adipocytes and prostate cancer cells with imaging FTIR microspectroscopy

Various epidemiological studies show a positive correlation between high intake of dietary FAs and metastatic prostate cancer (CaP). Moreover, CaP metastasizes to the bone marrow, which harbors a rich source of lipids stored within adipocytes. Here, we use Fourier transform infrared (FTIR) microspectroscopy to study adipocyte biochemistry and to demonstrate that PC-3 cells uptake isotopically labeled FA [deuterated palmitic acid (D(31)-PA)] from an adipocyte. Using this vibrational spectroscopic technique, we detected subcellular locations in a single adipocyte enriched with D(31)-PA using the upsilon(as+s)(C-D)(2+3) (D(31)-PA): upsilon(as+s)(C-H)(2+3) (lipid hydrocarbon) signal. In addition, larger adipocytes were found to consist of a higher percentage of D(31)-PA of the total lipid found within the adipocyte. Following background subtraction, the upsilon(as)(C-D)(2+3) signal illuminated starved PC-3 cells cocultured with D(31)-PA-loaded adipocytes, indicating translocation of the labeled FA. This study demonstrates lipid-specific translocation between adipocytes and tumor cells and the use of FTIR microspectroscopy to characterize various biomolecular features of a single adipocyte without the requirement for cell isolation and lipid extraction.

Differential Interactions between Transforming Growth Factor-β3/TβR1, TAB1, and CD2AP Disrupt Blood-Testis Barrier and Sertoli-Germ Cell Adhesion

The biochemical basis that regulates the timely and selective opening of the blood-testis barrier (BTB) to migrating preleptotene/leptotene spermatocytes at stage VIII of the epithelial cycle in adult rat testes is virtually unknown. Recent studies have shown that cytokines (e.g. transforming growth factor (TGF)-beta3) may play a crucial role in this event. However, much of this information relies on the use of toxicants (e.g. CdCl(2)), making it difficult to relay these findings to normal testicular physiology. Here we report that overexpression of TGF-beta3 in primary Sertoli cells cultured in vitro indeed perturbed the tight junction (TJ) barrier with a concomitant decline in the production of BTB constituent proteins as follows: occludin, N-cadherin, and ZO-1. Additionally, local administration of TGF-beta3 to testes in vivo was shown to reversibly perturb the BTB integrity and Sertoli-germ cell adhesion via the p38 MAPK and ERK signaling pathways. Most importantly, the simultaneous activation of p38 and ERK signaling pathways is dependent on the association of the TGF-beta3-TbetaR1 complex with adaptors TAB1 and CD2AP because if TbetaR1 was associated preferentially with CD2AP, only Sertoli-germ cell adhesion was perturbed without compromising the BTB. Collectively, these data illustrate that local production of TGF-beta3, and perhaps other TGF-betas and cytokines, by Sertoli and germ cells into the microenvironment at the BTB during spermatogenesis transiently perturbs the BTB and Sertoli-germ cell adhesion to facilitate germ cell migration when the activated TbetaRI interacts with adaptors TAB1 and CD2AP. However, TGF-beta3 selectively disrupts Sertoli-germ cell adhesion in the seminiferous epithelium to facilitate germ cell migration without compromising BTB when TbetaRI interacts only with adaptor CD2AP.

Ultrastructural characteristics of hemorrhagic, nonhemorrhagic, and recurrent cavernous malformations

Ultrastructural characteristics of hemorrhagic, nonhemorrhagic, primary, and recurrent central nervous system cavernous malformations (CMs) were examined in an attempt to clarify their pathological mechanisms. Thirteen specimens (nine from samples of CMs and four from healthy control tissue) were processed for ultrastructural study immediately after surgical or postmortem removal, by fixation in glutaraldehyde/formalin and postfixation in OsO4. Transmission electron microscopy was used to examine the vascular walls, endothelium, subendothelium, and cytoplasmic organelles. The vascular walls in CMs demonstrated abnormal ultrastructure with no basement membranes and astrocytic foot processes. Pericytes were rarely seen. Single-layer lining endothelial cells showed fenestrated luminal surfaces. Large gaps were observed at intercellular junctions between endothelial cells, and large vesicles with extremely thin plasma membranes bordering the lumen were common in the lesions that had previously hemorrhaged. Endothelial cells of recurrent CMs had more Weibel-Palade bodies, filopodia, cytoplasmic processes, micropinocytotic vesicles, and filaments than those in primary lesions and normal control tissues. The absence of the blood-brain barrier, normal supporting wall structure, and large vesicles bordering the lumen of CM vessels may explain leakage of red blood cells into surrounding brain in the absence of major hemorrhage. Proliferation of residual abnormal endothelial cells may contribute to the recurrence of surgically removed CMs.

Examination of the rat eye at the early stage of development with osmium tetroxide staining.

This communication describes the benefit of osmium tetroxide (OsO4) staining on the examination of the eye during the early stage of organogenesis of rat embryos. The embryos were obtained by laparotomy on embryonic day 12 (ED 12) and were stained with OsO4 for examination of the ocular tissues with a binocular stereo-microscope, light microscope and scanning electron microscope. At the binocular stereo-microscopic level, the invaginated lens placode, lens pit and optic cup were clearly distinguished. The osmium-stained lens placode and the optic cup were light brown and dark brown in color, respectively. Light microscopic examination revealed that OsO4 postfixation could provide superior paraffin-embedded embryonic sections. Scanning electron microscopic examination revealed the lens pit as a round opening between the lateral nasal prominence and maxillary prominence. Thus, a rapid technique by which the ocular tissues of rat embryos can be examined under a binocular stereo-microscope was developed. This OsO4 staining method will provide a useful tool for research on organogenesis and ocular development.

Avoiding Etching of Silver When Using Silver Staining With OsO4 Fixation

Abstract Post-fixing with osmium tetroxide before silver-staining sections causes problems because the OsCU can etch the deposited silver. However, it is not a good idea to perform silver enhancement after osmium tetroxide treatment because the deposited osmium can also act as a nucleating site for silver deposition, and the osmicated regions of the sample will be silver enhanced. If gold-toning is used after OsO4 post-fixation, the OSO4 can also nucleate gold particles. OsO4 should always come after silver enhancement.

Ultrastructural Osteopontin Localization in Papillary Stones Induced in Rats

To detect in situ the precise osteopontin (OPN) localization in papillary stones. Immunocytochemical labelling procedures are applied to detect OPN localizations in crystalline material of renal papillary stones. The tissue-processing procedure for electron microscopy, which includes OsO4 postfixation, preserves both immunocytochemical OPN reactivity and cellular membrane contrast up to the ultrathin section. Reflection-contrast light microscopical images are correlated with high resolution transmission-electron microscopical observations from consecutive ultrathin epon sections. Preserved crystalline material in interstitial and peripheral papillary stones is recognized as calcium oxalate monohydrate. After section incubation with markers conjugated to an antibody against OPN (alpha OPN) the crystals are converted into ghosts. In the ghosts, alpha OPN markers are present around microcrystals. The size of these microcrystals ranges from several nanometers to micrometers. It is observed (due to the OsO4-preserved membranes) that interstitial cells are separated from the stone surfaces by unidentified extracellular material, also present in the center as a stone matrix. The microcrystal-growth inhibitor OPN is detected in situ in interstitial stones induced in the rat's papilla and at the surface of the papilla.

Rapid microwave processing of skeletal muscle for TEM

Rapid tissue processing for transmission electron microscopy is a desired goal in a clinical EM laboratory. Reducing the time from tissue submission to examination in the electron microscope shortens turnaround time for diagnosis. Microwave enhanced tissue processing will accomplish these goals.Microwave technology reduces the time for tissue fixation and processing and may improve overall results. Fixation times can be reduced from minutes to seconds and resin polymerization from hours to minutes. Dehydration and infiltration steps can also be reduced, so that microwave rapid tissue processing can yield ultrathin sections from unfixed tissue in three hours.Three microwave protocols to process skeletal muscle biopsies are compared to standard processing. The protocols test the efficacy of microwave fixation, dehydration, and resin infiltration and polymerization. Protocol #1 uses microwave enhanced 4:1 formaldehyde/glutaraldehyde primary fixation followed by routine processing for remaining steps. Protocol #2 uses microwave primary fixation and 2% OsO4 post-fixation followed by routine processing. Protocol #3 uses microwave enhanced processing for entire procedure. Protocol #4 uses the standard processing technique.

Further observations on the morphological alterations of the glomerular capillary wall of the rat kidney caused by chemical and physical agents: standard procedures versus quick-freezing and freeze-substitution

The purpose of the present study was to pin-point which of the various preparatory steps required by different histological procedures (conventional, cryosubstitution, freezing followed by cryosubstitution or replication) induces alterations in the fine structure of the endothelial and epithelial surface coat (SC) and of the interposed glomerular basement membrane (GBM). Samples of rat kidney cortex prefixed by vascular perfusion, and isolated glomeruli fixed by immersion, were used. The results demonstrate a continuous, homogeneous and amorphous SC and a GBM devoid of laminae rarae when freezing is used and followed either by cryosubstitution and embedding or by fracture, deep etching and replication. Postfixation in OsO4, and especially dehydration in organic solvents at room temperature, generate a filamentous SC and a GBM with laminae rarae. The different morphology may be due to an extraction of glycoconjugates from SC and GBM during postosmication and/or especially to precipitation of these components during dehydration by organic solvents at room temperature. Studies on isolated glomeruli show comparable results and, in addition, demonstrate that the distance of the capillary loop from the surface of the block influences the morphology of SC and GBM. Rinsing the vascular bed before isolation does not induce a loss of SC or GBM components.

Localization of Ca(2+)-stores and tissue compartments with a Ca(2+)-binding capacity in the organ of Corti of the guinea-pig by electron energy-loss spectroscopy.

The addition of 10 mM CaCl2 to glutaraldehyde fixative leads to the formation of small electron-dense deposits in the organ of Corti of the guinea-pig. These precipitates are mainly attached to cell membranes in contact with different extracellular lymphatic fluids. A higher number of precipitates is localized in the acellular parts of tectorial and basilar membrane. Electron energy-loss spectroscopy (EELS) was used to determine the elemental composition of the deposits formed. The spectra showed a prominent signal at the Ca2+ L2,3 ionization edge. Oxygen could also be detected in all the precipitates analysed. EELS analysis of mitochondria of the inner and outer hair cells after conventional fixation (glutaraldehyde followed by post-fixation in OsO4) revealed a small but significant calcium signal.

Pituitary Secretory Granule Topography: Influence of Different Electron Microscopic Preparation Procedures on the Surface of Epon Sections

Human, rat and mouse pituitary tissues have been examined electron microscopically in transmission (TEM), scanning-transmission (STEM) and scanning (SEM) modes for the surface appearance of the secretory granules in tissue sections. Cryofixed and cryosectioned tissue showed only slightly protruding granule profiles which had a smooth surface. Cryofixed, freeze-dried and Epon embedded pituitaries, on the other hand, demonstrated swollen and furrowed surfaces over the granules after contact with water. This topography could also be seen after glutaraldehyde fixation but less after post-fixation in OsO4. The surface alterations in the sections of pituitary secretory granules are thought to be due to differences in the homogeneity of the resin infiltration, leaving resin-free openings where water can enter. It also seems probable that the Epon resin is more influenced by water than has been previously assumed, based on the findings of efficient elimination of osmium from the granules after incubation of tissue sections in water for only 10 min.

Microtubule number increases locally within regions of axonal compression

Along the length of myelinated axons, stereotyped variations in the interaction with the enveloping Schwann cell produce different amounts of compressive force on the axon and its internal elements. Comparisons of histologically distinct axonal regions indicate that compression of the axon by the Schwann cell increases in the following order: internodal regions of compact myelin < Schmidt-Lanterman (S-L) clefts < nodes of Ranvier. Quantitative morphometry has shown that regions of greater Schwann cell compression are associated with regions of smaller axon diameter and greater neurofilament (NF) packing density. By locally decreasing axon diameter and increasing NF density, Schwann cell compression (especially at the node of Ranvier) may slow the transport of those axonal structures - such as microtubules (MTs) - that move by a polymer sliding mechanism. Under this hypothesis, a local slowing of MT transport should produce a local increase in MT number, and here we have studied whether such a local increase in MT number can be quantitatively documented.A twenty-eight day old chicken was perfused by cardiac puncture with 4 1 of fixative (4% paraformaldehyde, 2% glutaraldehyde, 0.005% CaCl2, in 0.1M cacodylate buffer, pH 7.4). The somatic motor branch of the oculomotor nerve was removed (noting the proximo-distal orientation) and fixed overnight at 4°C, followed by a buffer rinse in 0.1M cacodylate buffer at room temperature and postfixation in OSO4. The nerve was then dehydrated in a graded ethanol series and embedded in PolyBed/812. Starting at the proximal end of the nerve segment, l00nm sections were cut at consecutive 2-3 um intervals over a 225 um length of nerve, stained with an LKB Ultrostainer, and photographed on a JEOL 100CX microscope. We selected two axons with normal morphology, in which the cytoskeletal polymer profiles were consistently cross-sectioned and in which nodes were located approximately 120 um distal to the first section taken. All sections from these 2 axons were photographed, and MTs were counted on 58,000X micrographs.

Applicability of using acrylic resins in post-embedding ultrastructural immunolabelling of human neutrophil granule proteins

In this study, quantitative assessments were carried out, (1) by light microscopy during tissue preparation for electron microscopy and (2) by electron microscopy after on-grid immunogold staining, to determine the suitability of using LR White and Lowicryl K4M thin sections to identify lactoferrin and elastase in the granules of human neutrophil leucocytes. Quantitative assessment of the effect of fixation, dehydration and embedding on the preservation of antigenicity during tissue preparation for electron microscopy, using light microscopic peroxidase anti-peroxidase immunocytochemistry, enabled the selection of preparation conditions that adequately preserved both antigenicity and ultrastructure. OsO4 post-fixation, following primary aldehyde fixation, improved the retention of antigenicity during dehydration and embedding and the preservation of fine structure. Partial rather than complete dehydration retained more of the antigenicity. The efficiency, sensitivity and resolution of immunolabelling and the ultrastructure and quality of sections achieved after embedding in LR White were superior to those obtained after embedding in Lowicryl K4M. Consequently room temperature embedding in LR White following double fixation and partial dehydration is a better and more reliable preparation technique than low-temperature embedding in Lowicryl K4M following single fixation and partial dehydration for localizing lactoferrin and elastase to the specific and primary granules respectively in human neutrophilic granulocytes by the on-grid immunogold staining method.

Ultrastructural electron probe X-ray microanalytical reaction product identification of three different enzymes in the same mouse resident peritoneal macrophage.

Simultaneous cytochemical enzyme localization procedures for peroxidase (PO) plus acid phosphatase (AcP-ase) and/or aryl sulphatase (AS) have been investigated at the ultrastructural (EM) level. Electron probe X-ray microanalysis (EPMA) will identify and differentiate the reaction products. Dual reaction product localization of PO plus AcP-ase or alternatively PO plus AS have been obtained in the same mouse resident peritoneal macrophage. This has been acquired by first performing a PO-reaction followed by AcP-ase or followed by AS. In both cases PO-related reaction products (PODAB/Os or PODAB/Pt) were localized in nuclear envelope (NE) and rough endoplasmic reticulum (RER). Cells were identified by this reaction product as resident macrophages. Reaction products from the AcP-ase related cerium (AcP-aseCe), localized in lysosomes have been identified and differentiated from the PO-related osmium containing products. Similarly AS related barium (ASBa), localized in lysosomal structures and (R)ER was identified and differentiated. Triple reaction product localization of PO followed by AcP-ase plus AS could also be obtained. In this case, PO-related platinum containing reaction products (PODAB/Pt or PODAB/Os) in NE and RER has been identified and differentiated from the AcP-ase related lysosomal cerium (AcP-aseCe) and the AS related barium localized in lysosomal and (R)ER structures. Reversing the sequences in both dual cytochemical procedures: AcP-aseCe or ASBa followed by PODAB/Os (or PODAB/Pt) resulted in AcP-aseCe or ASBa activity related reaction products only. Reversing the sequence in the triple reaction procedures (ASBa followed by AcP-aseCe) resulted in the absence of the barium containing reaction products. By application of OsO4 postfixation with aminotriazole (ATR) additives the detrimental effects upon the various precipitates have been confirmed. In LM studies, using rat intestine and non-metal identification reactions for two of the enzymes (pararosaniline for AcP-ase, DAB for peroxidase), the influences of the metal ions used in EM were tested on the appearance of the coloured reaction products. Cerium ions used in EM for detection of AcP-aseCe activity have been shown to influence the PODAB visibility in LM and EM experiments. From the AS reaction media components neither barium ions nor p-nitro catachol sulphate influenced the LM visibility of the PO reaction.

Sites of leucine, arginine, and glycine accumulation in the absorptive trichomes of a carnivorous bromeliad

Ultrastructural localization of leucine, arginine, and glycine in the nutrient absorptive trichomes of the bromeliad Brocchinia reducta was achieved through a precipitation reaction with osmium tetroxide. Leaves incubated with amino acid solutions, then fixed with glutaraldehyde and postfixed with OsO4 exhibited within the trichomes site specific accumulations of an electron-dense precipitate which were absent in distilled water-incubated controls. Four regions of leucine accumulation occurred: (i) in the outer trichome walls, (ii) within mitochondrial cristae, (iii) on the surface of lipid bodies, and (iv) in tubular invaginations of the inner envelope membrane of plastids. Glycine-incubated leaves had dense deposits in the mitochondrial matrix. Arginine-treated tissue had deposits only within the outer trichome walls. The initial glutaraldehyde fixation stabilized accumulations of the amino acid by reacting with the primary amine groups to form a Schiff's base. Subsequent OsO4 postfixation was presumably oxidative with OsO4 being reduced to an insoluble form.

Ultrastructure of complex carbohydrates of rodent and monkey ependymal glycocalyx and meninges.

The surfaces of the brain offer metabolic and mechanical support to the underlying parenchyma. Mouse, rat, and monkey brains were fixed by immersion in a glutaraldehyde fixative or glutaraldehyde with cetylpyridinium chloride, followed by block staining for complex carbohydrates using alcian blue with OsO4 postfixation, or OsO4 postfixative solution containing ruthenium red, or alcian blue and then ruthenium red-OsO4 treatment. The ependyma in these species had a glycocalyx extending into the ventricular fluid as a finely filamentous network when stained with alcian blue or with alcian blue followed by ruthenium red-OsO4. Mice in the middle age range had stained material in this glycocalyx resembling the hyaluronic acid reported in the ocular vitreous body. Similar material was seen in the arachnoidal space of these mice and in the inner connective tissue matrix of the dura mater. Both the mouse and monkey had a cell-free zone, termed the inner dural matrix zone, between the thick fibrous dura and its innermost cellular layer. This zone contained filamentous and globular alcian blue-stained material. The complex carbohydrates of the mouse ependymal glycocalyx and inner dural matrix zone underwent changes developmentally. Aged rats were injected intraventricularly with latex beads, which, along with extravasated erythrocytes, were seen to adhere to the ependymal glycocalyx. A similar adhesion of erythrocytes was seen in the mouse and monkey ependymal glycocalyx and in the filamentous network of the mouse and monkey inner dural matrix zone. The ependymal glycocalyx, formed in part of complex carbohydrates, is much thicker than previously demonstrated. Some activities related to the ependymal lining of the ventricles, including the movement of cells or particles, the penetration of metabolites or serum-protein fractions (e.g., immunoglobulins), and cell-surface hydration, probably depend in part on complex carbohydrates that provide a sticky, electrically negative, hydrophilic environment. The complex carbohydrates in the inner dural matrix zone might provide mechanical buffering. Complex carbohydrates in the arachnoidal space may help to maintain a loose tissue that needs not only to be hydrated, but also to be open enough to provide cerebrospinal fluid circulation.