Phosphotungstic Acid As An Electron Stain

1968 ◽  
Vol 26 ◽  
pp. 36-37
Author(s):  
Daniel C. Pease
Keyword(s):  
Chondroitin Sulfate ◽  
Room Temperature ◽  
Phosphotungstic Acid ◽  
Freeze Substitution ◽  
Basement Membranes ◽  
Effective Electron ◽  
Tissue Sections ◽  
Brush Borders ◽  
Effects Of Ph ◽  
Acid Environment

Under certain conditions phosphotungstic acid (PTA) can be used as an effective electron stain for demonstrating polysaccharide moieties in tissue sections. There it selectively combines with the glycoproteins of basement membranes, the mucoid layers associated with brush borders, the mucus of goblet cells, the chondroitin sulfate of cartilage matrix, glyco-gen, and “glycocalyces” in general. The initial published investigation indicated that an acid environment was essential for this specificity, but the effect of pH was not systematically explored.Rib cartilage of newborn rats was chosen for a detailed study of the effects of pH on PTA staining, particularly to provide ancillary information on the formation, secretion and deposition of chondroitin sulfate. Cartilage was prepared without chemical fixation by “freeze-substitution” with 70% glycol, or by “inert dehydration” with glycol at room temperature. Dehydrated material was embedded in prepolymerized hydroxy-propyl methacrylate without an intermediate solvent.

scholarly journals Developmental changes in heparan sulfate expression: in situ detection with mAbs.

1992 ◽  
Vol 119 (4) ◽  
pp. 961-975 ◽  
Author(s):  
G David ◽  
X M Bai ◽  
B Van der Schueren ◽  
J J Cassiman ◽  
H Van den Berghe
Keyword(s):  
Heparan Sulfate ◽  
Chondroitin Sulfate ◽  
Regional Differences ◽  
Cell Types ◽  
Basement Membranes ◽  
Developmentally Regulated ◽  
Tissue Sections ◽  
Heparan Sulfates ◽  
In Situ Detection

Two mAbs that are specific for heparan sulfate-related epitopes have been raised and used to analyze the cellular and tissular distribution of this glycosaminoglycan during development. mAb 10E4 reacts with an epitope that occurs in native heparan sulfate chains and that is destroyed by N-desulfation of the glycosaminoglycan. The antibody does not react with hyaluronate, chondroitin sulfate, or DNA, and reacts only poorly with heparin. The reactivity of proteoglycan extracts or tissue sections with the 10E4 antibody is completely abolished by heparitinase, but is only partially affected by heparinase. mAb 3G10, in contrast, reacts only with heparitinase-treated heparan sulfate chains, proteoglycans, or tissue sections. The 3G10 epitope is destroyed by treatment with mercuric acetate, which indicates that the desaturated uronate generated by the lyase is essential for the reactivity of the antibody. The 3G10 epitope is not generated by treating heparan sulfate proteoglycans with heparinase or chondroitin sulfate proteoglycans with chondroitin sulfate lyases, which indicates that the 3G10 antibody recognizes desaturated uronates that occur in specific structural contexts. The antibody 10E4 and, after heparitinase treatment, the antibody 3G10 decorate the surfaces of many cell types and the extracellular matrix in proximity of the cells, in particular, the basement membranes. The analysis of embryonic and adult tissues reveals important temporal and regional differences in the abundance of the 10E4 and 3G10 epitopes at these sites. Moreover, the staining pattern of the two antibodies is not always superimposable, which is indicative of regional differences in the exposure or structure of the tissular heparan sulfates. As a whole the results suggest that heparan sulfate abounds at sites of active morphogenesis and that the expression of this glycosaminoglycan is developmentally regulated.

High-Resolution Scanning Electron Microscopy of Biological Specimens

1988 ◽  
Vol 46 ◽  
pp. 186-187
Author(s):  
M. Müller ◽  
R. Hermann
Keyword(s):  
High Resolution ◽  
Freeze Drying ◽  
Room Temperature ◽  
Structural Information ◽  
Freeze Substitution ◽  
Critical Point Drying ◽  
Sem Study ◽  
Major Factors ◽  
Structural Preservation ◽  
Preparation Techniques

Three major factors must be concomitantly assessed in order to extract relevant structural information from the surface of biological material at high resolution (2-3nm).Procedures based on chemical fixation and dehydration in graded solvent series seem inappropriate when aiming for TEM-like resolution. Cells inevitably shrink up to 30-70% of their initial volume during gehydration; important surface components e.g. glycoproteins may be lost. These problems may be circumvented by preparation techniques based on cryofixation. Freezedrying and freeze-substitution followed by critical point drying yields improved structural preservation in TEM. An appropriate preservation of dimensional integrity may be achieved by freeze-drying at - 85° C. The sample shrinks and may partially collapse as it is warmed to room temperature for subsequent SEM study. Observations at low temperatures are therefore a necessary prerequisite for high fidelity SEM. Compromises however have been unavoidable up until now. Aldehyde prefixation is frequently needed prior to freeze drying, rendering the sample resistant to treatment with distilled water.

Otoconia perfect/imperfect crystals

1994 ◽  
Vol 52 ◽  
pp. 42-43
Author(s):  
César D. Fermin ◽  
Dale Martin
Keyword(s):  
Image Processing ◽  
Room Temperature ◽  
Phosphotungstic Acid ◽  
Gallus Domesticus ◽  
Crystal Matrix ◽  
Organic Templates ◽  
Image Processing Algorithms ◽  
Processing Algorithms ◽  
Diffraction Patterns

Otoconia of higher vertebrates are interesting biological crystals that display the diffraction patterns of perfect crystals (e.g., calcite for birds and mammal) when intact, but fail to produce a regular crystallographic pattern when fixed. Image processing of the fixed crystal matrix, which resembles the organic templates of teeth and bone, failed to clarify a paradox of biomineralization described by Mann. Recently, we suggested that inner ear otoconia crystals contain growth plates that run in different directions, and that the arrangement of the plates may contribute to the turning angles seen at the hexagonal faces of the crystals.Using image processing algorithms described earlier, and Fourier Transform function (2FFT) of BioScan Optimas®, we evaluated the patterns in the packing of the otoconia fibrils of newly hatched chicks (Gallus domesticus) inner ears. Animals were fixed in situ by perfusion of 1% phosphotungstic acid (PTA) at room temperature through the left ventricle, after intraperitoneal Nembutal (35mg/Kg) deep anesthesia. Negatives were made with a Hitachi H-7100 TEM at 50K-400K magnifications. The negatives were then placed on a light box, where images were filtered and transferred to a 35 mm camera as described.

Ultrastructural aspects of olfactory signal transduction and its development

1994 ◽  
Vol 52 ◽  
pp. 142-143
Author(s):  
Bert Ph. M. Menco
Keyword(s):  
Signal Transduction ◽  
Olfactory Receptor ◽  
Receptor Cell ◽  
Freeze Substitution ◽  
Luminal Surface ◽  
Tissue Sections ◽  
Olfactory Receptor Cells ◽  
Receptor Cells ◽  
Olfactory Signal ◽  
Odor Molecule

Vertebrate olfactory receptor cells are specialized neurons that have numerous long tapering cilia. The distal parts of these cilia line the interface between the external odorous environment and the luminal surface of the olfactory epithelium. The length and number of these cilia results in a large surface area that presumably increases the chance that an odor molecule will meet a receptor cell. Advanced methods of cryoprepration and immuno-gold labeling were particularly useful to preserve the delicate ultrastructural and immunocytochemical features of olfactory cilia required for localization of molecules involved in olfactory signal-transduction. We subjected olfactory tissues to freeze-substitution in acetone (unfixed tissues) or methanol (fixed tissues) followed by low temperature embedding in Lowicryl K11M for that purpose. Tissue sections were immunoreacted with several antibodies against proteins that are presumably important in olfactory signal-transduction.

High-pressure freezing and freeze substitution of teliospores of the rust fungus Gymnosporangium clavipes

1990 ◽  
Vol 48 (3) ◽  
pp. 692-693
Author(s):  
Robert W. Roberson
Keyword(s):  
High Pressure ◽  
Room Temperature ◽  
Pathogenic Fungus ◽  
Rust Fungus ◽  
Freeze Substitution ◽  
Ice Crystals ◽  
High Pressure Freezing ◽  
Thin Walled Structures ◽  
Cytological Changes ◽  
Dextran Solution

The use of cryo-techniques for the preparation of biological specimens in electron microscopy has led to superior preservation of ultrastructural detail. Although these techniques have obvious advantages, a critical limitation is that only 10-40 μm thick cells and tissue layers can be frozen without the formation of distorting ice crystals. However, thicker samples (600 μm) may be frozen well by rapid freezing under high-pressure (2,100 bar). To date, most work using cryo-techniques on fungi have been confined to examining small, thin-walled structures. High-pressure freezing and freeze substitution are used here to analysis pre-germination stages of specialized, sexual spores (teliospores) of the plant pathogenic fungus Gymnosporangium clavipes C & P.Dormant teliospores were incubated in drops of water at room temperature (25°C) to break dormancy and stimulate germination. Spores were collected at approximately 30 min intervals after hydration so that early cytological changes associated with spore germination could be monitored. Prior to high-pressure freezing, the samples were incubated for 5-10 min in a 20% dextran solution for added cryoprotection during freezing. Forty to 50 spores were placed in specimen cups and holders and immediately frozen at high pressure using the Balzers HPM 010 apparatus.

A combined pseudoreplica-immunocytochemical technique for research and diagnostic virology

1990 ◽  
Vol 48 (3) ◽  
pp. 900-901
Author(s):  
Blayne Fritz ◽  
Stanley J. Naides ◽  
Kenneth Moore
Keyword(s):  
Phosphotungstic Acid ◽  
Immunogold Labelling ◽  
Uranyl Acetate ◽  
Distilled Water ◽  
Clinical Specimens ◽  
Tissue Sections ◽  
Specimen Retrieval ◽  
Transmission Electron ◽  
Viral Particles ◽  
Diagnostic Virology

The pseudoreplica method of staining viral particles for visualization by transmission electron microscopy is a very popular technique. The ability to concentrate clinical specimens while semi-embedding viral particles makes it especially well suited for morphologic and diagnostic virology. Immunolabelling viral particles with colloidal gold is a technique frequently employed by both research and diagnostic virologists. We have characterized a procedure which provides the advantage of both by modifying and combining pseudoreplica staining and immunogold labelling.Modification of specimen retrieval and delay of staining allows us to utilize pseudoreplica processed specimens within our standard immunogold labelling protocol. In brief, we absorbed samples onto 2% agarose, added.25% Formvar and wicked dry. We then floated the Formvar-virus film onto double distilled water, added grids and retrieved with parafilm. The Formvar-virus specimens were then treated as thin tissue sections within our standard two stage immunolabelling protocol. Following completion of immunogold labelling; each grid was negatively stained with phosphotungstic acid or uranyl acetate contrast stains.

Ultrastructural and immunocytochemical studies of the rat hypothalamo-neurohypophysial system processed by rapid freezing and freeze-substitution fixation

1990 ◽  
Vol 48 (3) ◽  
pp. 884-885
Author(s):  
Seiji Shioda ◽  
Yasumitsu Nakai ◽  
Atsushi Ichikawa ◽  
Hidehiko Ochiai ◽  
Nobuko Naito
Keyword(s):  
Osmium Tetroxide ◽  
Freeze Substitution ◽  
Ultrastructural Localization ◽  
Wistar Rat ◽  
Neurosecretory Cells ◽  
Rapid Freezing ◽  
Sodium Metaperiodate ◽  
Tissue Sections ◽  
Ultrathin Sections ◽  
Electron Microscopes

The ultrastructure of neurosecretory cells and glia cells in the supraoptic nucleus (SON) of the hypothalamus and the neurohypophysis (PN) was studied after rapid freezing followed by substituion fixation. Also, the ultrastructural localization of vasopressin (VP) or its carrier protein neurophys in II (NPII) in the SON and PN was demonstrated by using a post-embedding immunoco1loidal gold staining method on the tissue sections processed by rapid freezing and freeze-substitution fixation.Adult male Wistar rat hypothalamus and pituitary gland were quenched by smashing against a copper block surface precooled with liquid helium and freeze-substituted in 3% osmium tetroxide-acetone solutions kept at -80°C for 36-48h. After substituion fixation, the tissue blocks were warmed up to room temperature, washed in acetone and then embedded in an Epon-Araldite mixture. Ultrathin sections mounted on 200 mesh nickel grids were immersed in saturated sodium metaperiodate and then incubated in each of the following solutions: 1 % egg albumin in phosphate buffer, VP or NPII (1/1000-1/5000) antiserum 24h at 4°C, 3) colloidal gold solution (1/20) 1h at 20°C. The sections were washed with distilled waterand dried, then stained with uranylacetate and lead citrate and examined with Hitachi HU-12A and H-800 electron microscopes.

Characterisation of an epithelium-like layer of cells in the multicellular Dictyostelium discoideum slug

1993 ◽  
Vol 105 (1) ◽  
pp. 243-253
Author(s):  
M. Fuchs ◽  
M.K. Jones ◽  
K.L. Williams
Keyword(s):  
Dictyostelium Discoideum ◽  
Intercellular Space ◽  
Room Temperature ◽  
Cell Layer ◽  
Freeze Substitution ◽  
Specific Cell ◽  
Peripheral Layer ◽  
Apical Membranes ◽  
Multicellular Organisms ◽  
Peripheral Cells

Ultrarapid freezing (RF) followed by freeze-substitution (FS) provide superior preservation of the Dictyostelium discoideum multicellular slug tissue over conventional methods of chemical fixation at room temperature. The peripheral cells of slugs prepared by RF and FS form a tight layer of flattened cells. This cell layer resembles epithelia of other multicellular organisms in that it has close junctional contact between cells associated with the extracellular matrix (ECM, slime sheath). This is the first report that clearly demonstrates the existence of such peripheral cellular specialisation in this otherwise well-studied model system. Junctional contacts between adjacent cells mean that there is no intercellular space evident between apical membranes of apposing cells, and basally the intermembraneous space between peripheral cells is less than 10 nm. By contrast, the intercellular space between internal cells is approximately 10–25 nm. The shape of the peripheral cells varies with their location around the slug. In the posterior prespore zone, the peripheral cells are squamous and exhibit polarity along their antero-posterior axis. In the anterior prestalk zone, peripheral cells are less flattened, project irregular filipodia between internal cells, and are polarised along their apical-basal axis. Colloidal gold immunocytochemistry with the markers MUD1, MUD50 and MUD62 demonstrated that the peripheral layer is formed of prestalk cells in the anterior region and ventrum, and mostly prespore cells along the dorsum. Thus, the peripheral layer, while having specific cell classes in different regions, is not differentiation-specific. Rather, it appears that the structure of these epithelium-like cells is influenced by interaction with molecules of the ECM (sheath).

scholarly journals cDNA Cloning of the Basement Membrane Chondroitin Sulfate Proteoglycan Core Protein, Bamacan: A Five Domain Structure Including Coiled-Coil Motifs

1997 ◽  
Vol 136 (2) ◽  
pp. 433-444 ◽  
Author(s):  
Rong-Rong Wu ◽  
John R. Couchman
Keyword(s):  
Basement Membrane ◽  
Chondroitin Sulfate ◽  
Core Protein ◽  
Coiled Coil ◽  
Basement Membranes ◽  
Cdna Clones ◽  
Molecular Architecture ◽  
Unusual Structure ◽  
Extracellular Matrix Molecule

Basement membranes contain several proteoglycans, and those bearing heparan sulfate glycosaminoglycans such as perlecan and agrin usually predominate. Most mammalian basement membranes also contain chondroitin sulfate, and a core protein, bamacan, has been partially characterized. We have now obtained cDNA clones encoding the entire bamacan core protein of Mr = 138 kD, which reveal a five domain, head-rod-tail configuration. The head and tail are potentially globular, while the central large rod probably forms coiled-coil structures, with one large central and several very short interruptions. This molecular architecture is novel for an extracellular matrix molecule, but it resembles that of a group of intracellular proteins, including some proposed to stabilize the mitotic chromosome scaffold. We have previously proposed a similar stabilizing role for bamacan in the basement membrane matrix. The protein sequence has low overall homology, apart from very small NH2- and COOH-terminal motifs. At the junctions between the distal globular domains and the coiled-coil regions lie glycosylation sites, with up to three N-linked oligosaccharides and probably three chondroitin chains. Three other Ser-Gly dipeptides are unfavorable for substitution. Fusion protein antibodies stained basement membranes in a pattern commensurate with bamacan, and they also Western blotted bamacan core protein from rat L2 cell cultures. The antibodies could also specifically immunoprecipitate an in vitro transcription/translation product from a full-length bamacan cDNA. The unusual structure of this proteoglycan is indicative of specific functional roles in basement membrane physiology, commensurate with its distinct expression in development and changes in disease models.

scholarly journals Phosphotungstic acid as a novel acidic catalyst for carbohydrate protection and glycosylation

RSC Advances ◽  
2019 ◽  
Vol 9 (58) ◽  
pp. 33853-33862
Author(s):  
Jyun-Siao Chen ◽  
Arumugam Sankar ◽  
Yi-Jyun Lin ◽  
Po-Hsun Huang ◽  
Chih-Hsiang Liao ◽  
...  
Keyword(s):  
Room Temperature ◽  
Phosphotungstic Acid ◽  
Carbohydrate Chemistry ◽  
Acidic Catalyst ◽  
Primary Reactions

This work demonstrates the utilization of PTA as an acidic catalyst in primary reactions of carbohydrate. PTA can be widely used as a catalyst for carbohydrate chemistry. The reactions are executed at 0 °C or room temperature in good yields.

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