THE PREFERENTIAL DEMONSTRATION OF DEOXYRIBONUCLEIC ACID BY A PERIODIC ACID-SILVER DIAMINE METHOD FOR LIGHT AND ELECTRON MICROSCOPY

Lungs from marsupials, bats and rodents were studied by light and electron microscopy. In all three groups, the great alveolar cells exhibit similar morphologic and cytochemical characteristics. Cytoplasmic vacuoles seen in these cells by light microscopy correspond to cytosomes that are demonstrable in them by electron microscopy. Such cytosomes are osmiophilic, periodic acid-Schiff-positive and stainable with Sudan black after acetone extraction. After fixation in a mixture of aldehydes, followed by extraction in chloroform-methanol and postfixation in osmium tetroxide, cytosomes lose their osmiophilia. The cytoplasm of the great alveolar cell is notable for a loosely ordered granular endoplasmic reticulum, an extensive Golgi apparatus and numerous multivesicular bodies. Many forms transitional in appearance between multivesicular bodies and cytosomes are present. In these, osmiophilic matter occupies the intervesicular space. It is proposed that these bodies are the precursors of cytosomes. The cytosomes are interpreted to be products of the "lysosomal" system in this cell. Ultimately they are secreted onto the alveolar surface.

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Effects of O3 on submucosal gland of bonnet monkey trachea

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100076962 ◽

1983 ◽

Vol 41 ◽

pp. 678-679

Author(s):

S. Yamashiro ◽

D. Wilson ◽

J. St. George ◽

D. Hyde ◽

C. Plopper ◽

...

Keyword(s):

Electron Microscopy ◽

Periodic Acid ◽

Light And Electron Microscopy ◽

Lung Parenchyma ◽

Uranyl Acetate ◽

Upper Airways ◽

Periodic Acid Schiff ◽

Transmission Electron ◽

Bonnet Monkey ◽

Submucosal Glands

In the past, ozone inhalation studies have focused on the lower airways and lung parenchyma. The purpose of this study was to evaluate the effects of ozone on submucosal glands of upper airways. Six adult male bonnet monkeys were exposed to 0.64 ppm ozone continuously for 7 days, and three were exposed to chamber conditions without ozone. The animals were exsanguinated under barbiturate anesthesia. The trachea and lung were fixed by airway infusion of Karnovsky's fixative, which was adjusted to pH 7.4 and 440 milliOsmols. Sagittal sections of ventral trachea were embedded in glycol methacrylate and Araldite 502 for light and electron microscopy. One micrometer methacrylate sections were stained with Alcian blue-periodic acid Schiff (AB/PAS). Selected areas of Araldite-embedded tissue were sectioned for transmission electron microscopy, stained with uranyl acetate and lead citrate and examined with a Zeiss EM 10. Volume percentages of the lumen, granular and nongranular regions of fhe gland and the duct wall, respectively, were estimated by stereologic methods on AB/PAS stained sections.

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Periodic acid-thiocarbohydrazide-silver methenamine(PATS) reaction: An improved silver methodology for light and electron microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100111434 ◽

1984 ◽

Vol 42 ◽

pp. 264-265 ◽

Cited By ~ 1

Author(s):

B. Giammara ◽

T. Romaine ◽

W. Ambrose ◽

J. Hanker

Keyword(s):

Electron Microscopy ◽

Osmium Tetroxide ◽

Periodic Acid ◽

Patent Application ◽

Light And Electron Microscopy ◽

Basement Membranes ◽

Full Description ◽

Periodic Acid Schiff ◽

Reticular Fibers ◽

Pas Reaction

Many variations of the periodic acid-Schiff(PAS) reaction have been utilized for electron microscopy based on the Gomori periodic acid-silver methenamine reaction (1) or the periodic acid-thiocarbohydrazide-osmium tetroxide(PATCO) reaction (2,3). These reactions are widely employed and have been very useful for the demonstration of one or more biomacromolecules or structures such as glycogen, basement membranes, reticular fibers or lipopolysaccharide. However, these reactions have various drawbacks such as complexity of methodology, ability to stain only a limited number of these components, or lack of adaptability for both light and electron microscopy. Our newly devised PATS reaction is relatively easy to perform. A full description of the details must await the outcome of a pending patent application. It consists essentially of a stepwise treatment of the sample with periodic acid, thiocarbohydrazide(TCH) and silver methenamine.

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Light and electron microscopic demonstration by the PATS reaction of peripheral nerve regeneration

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100156742 ◽

1989 ◽

Vol 47 ◽

pp. 952-953 ◽

Cited By ~ 1

Author(s):

B. Giammara ◽

E. Anderson ◽

P. Yates ◽

J. Hanker

Keyword(s):

Electron Microscopy ◽

Toluidine Blue ◽

Periodic Acid ◽

Light And Electron Microscopy ◽

Nerve Fibers ◽

Blue Staining ◽

Hydroxyl Groups ◽

Distal Stump ◽

Thin Sections ◽

Toluidine Blue Staining

Although periodic acid-Schiff(PAS) type reactions have been applied to nervous tissues for many years, interest has centered upon staining glycolipids, principally myelin constituents such as the class of sphingolipids. The staining of these compounds such as sphingomyelin has generally been attributed to the presence of amino and hydroxyl groups on adjacent carbon atoms of carbohydrate of the sphingosine moiety. But unsaturated lipids also give the reaction and sphingolipids stain even if carbohydrate moieties are absent. This reaction has been used for staining myelin sheaths but lipid solvents must be avoided in processing the specimens. Toluidine blue staining of semi-thin sections of epoxy- embedded nerve specimens has also been widely used to study regenerating fibers after nerve transection or avulsion. A recent study was made in our laboratories of conduits (sleeves) tailored from biodegradable polyester (VicrylR) mesh to guide the reconnection of regenerating fibers from the proximal stump of a rat sciatic nerve, across an 11 mm gap, with fibers in the distal stump of the interrupted nerve. Complete reconnection of the stumps was observed as early as one month after creating the avulsive nerve injury.Comparison of transverse sections of the repaired sciatic with sections of control nerve with the toluidine blue stain, however, showed little evidence of axonal regeneration after one month (Figs. 1,3). A variation of the PAS reaction (depositing silver) for light and electron microscopy developed in our laboratories (PATS reaction, 5) was than applied to the study of the semi-thin sections of the epoxy-embedded control and repaired sciatic nerves of the same rat one month postsurgery. Correlative light and scanning electron microscopy by SEI and BEI modes could then be performed since the PATS reaction produced very satisfactory staining of the semi-thin sections (Figs. 3-5). Myelin was not stained by the PATS reaction in these specimens since the nerves had been processed with lipid solvents for epoxy embedment. Schwann cells, however, were very prominent in control but not in the repaired nerve. The inner layers of endoneurium and all pericapillaries associated with nerve fibers were intensely stained due to their reticulin content in both control and repaired nerve (Figs. 2,4). This was not unexpected because the PATS reaction employs a silver methenamine reagent. Thus, with the PATS reaction axons could be identified in sections of repaired nerve (Fig. 4) that could not be discerned with toluidine blue staining (Fig. 3). In sections of repaired nerve stained with either toluidine blue or the PATS reaction few axons or axis cylinders were observed but more were seen with the PATS stain (Figs. 3,4). In control nerve sections stained with either procedure many were seen (Figs. 1,2).

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Positive stain for light and electron microscopic demonstration of spirochetes in subgingival plaque and crevicular fluid samples from periodontal diseased sites

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100157395 ◽

1989 ◽

Vol 47 ◽

pp. 1082-1083 ◽

Cited By ~ 1

Author(s):

B. Giammara ◽

E.J. Burkes ◽

R. Scruggs ◽

G. Greco ◽

P. Yates ◽

...

Keyword(s):

Electron Microscopy ◽

Periodic Acid ◽

Light And Electron Microscopy ◽

Subgingival Plaque ◽

Microbial Count ◽

Electron Microscopic ◽

Microbial Composition ◽

Periodic Acid Schiff ◽

Clear Cut ◽

Crevicular Fluid

In a recent study of 400 subgingival plaque samples from over 110 adult periodontitis patients, spirochetes were the overwhelming microbial type, averaging about 45% of the microbial count. This finding supports earlier arguments that spirochetes are pathognomonic in periodontal disease. Other studies had shown clear-cut differences in the microbial composition of healthy and diseased subgingival sites — the proportion of spirochetes being significantly higher in the latter. Another study indicated that periodontal deterioration at these sites could be predicted better by increased proportions of motile rods and spirochetes than by clinical measurements. However, spirochetes of all sizes and species do not show the same degree of association with periodontal breakdown. Moreover, spirochetes are usually difficult to culture and stain; they are generally monitored by darkfield or phase contrast microscopy.The PATS reaction, a modified periodic acid-Schiff(PAS) reaction which deposits silver for light and electron microscopy appears to stain Gram(-) bacteria positively as well as neutrophils and activated macrophages. When studying the stained Gram(-) bacteria on coverslip smears of subgingival plaque or crevicular fluid samples of patients by light microscopy, varying numbers of intensely stained spirochetes of different sizes were observed (Figs. 1,2). More spirochetes were usually seen in samples from diseased sites. After drying replicate PATS-stained coverslips with hexamethyldisilazane they were sputter coated with gold, and. then examined by the SEI and BEI modes of scanning electron microscopy (Figs. 3-6). A permanent record of the proportions of large, medium and small spirochetes at each site could thus be obtained. Generally, greater numbers of gram negative bacteria including some spirochetes were stained in samples from diseased sites. At some sites, however, spirochetes were the predominant microbes in both crevicular fluid and subgingival plaque (Fig. 1).

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Three-dimensional electron microscopy for endothelial glycocalyx observation using Alcian blue with silver enhancement

Medical Molecular Morphology ◽

10.1007/s00795-020-00267-1 ◽

2020 ◽

Author(s):

Shumpei Mukai ◽

Takashi Takaki ◽

Tasuku Nagumo ◽

Mariko Sano ◽

Dedong Kang ◽

...

Keyword(s):

Electron Microscopy ◽

Alcian Blue ◽

Periodic Acid ◽

Light And Electron Microscopy ◽

Three Dimensional ◽

Kidney Tissue ◽

Endothelial Glycocalyx ◽

Simple Method ◽

Ffpe Samples ◽

Formalin Fixed

Abstract Glycocalyx (GCX) is a thin layer of negatively charged glycoproteins that covers the vascular endothelial surface and regulates various biological processes. Because of the delicate and fragile properties of this structure, it is difficult to detect GCX morphologically. We established a simple method for a three-dimensional visualization of endothelial GCX using low-vacuum scanning electron microscopy (LVSEM) on formalin-fixed paraffin-embedded (FFPE) sections. Mouse kidney tissue was fixed with 10% buffered formalin containing 1% Alcian blue (ALB) via perfusion and immersion. FFPE sections were observed by light microscopy (LM) and LVSEM, and formalin-fixed epoxy resin-embedded ultrathin sections were observed by transmission electron microscopy (TEM). The endothelial GCX from various levels of kidney blood vessels was stained blue in LM and confirmed as a thin osmiophilic layer in TEM. In LVSEM, the sections stained by periodic acid methenamine silver (PAM) revealed the endothelial GCX as a layer of dense silver-enhanced particles, in both the samples fixed via perfusion and immersion. Correlative light and electron microscopy (CLEM) revealed the fine visible structure of endothelial GCX. This simple method using FFPE samples with ALB will enable the three-dimensional evaluation of endothelial GCX alterations in various human diseases associated with endothelial injury in future studies.

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SPERMIOGENESIS IN CANCER CRABS

The Journal of Cell Biology ◽

10.1083/jcb.43.3.575 ◽

1969 ◽

Vol 43 (3) ◽

pp. 575-603 ◽

Cited By ~ 85

Author(s):

Susan G. Langreth

Keyword(s):

Electron Microscopy ◽

Endoplasmic Reticulum ◽

Nucleic Acid ◽

Sperm Cell ◽

Periodic Acid ◽

Light And Electron Microscopy ◽

Mature Sperm ◽

Periodic Acid Schiff ◽

The Core ◽

Inner Region

Spermiogenesis in Cancer crabs was studied by light and electron microscopy. The sperm are aflagellate, and when mature consist primarily of a spherical acrosome surrounded by the nucleus with its short radiating arms. The acrosome forms by a coalescence of periodic acid-Schiff-positive (PAS-positive) vesicles. During spermiogenesis one edge of the acrosomal vesicle invaginates to form a PAS-negative central core. The inner region of the acrosome bounding the core contains basic proteins which are not complexed to nucleic acid. The formation of an elaborate lattice-like complex of fused membranes, principally from membranes of the endoplasmic reticulum, is described. These membranes are later taken into the nucleus and subsequently degenerate. In late spermatids, when most of the cytoplasm is sloughed, the nuclear envelope and the cell membrane apparently fuse to become the limiting boundary over most of the sperm cell. In the mature sperm the chromatin of the nucleus and arms, which is Feulgen-positive, contains no detectable protein. The chromatin filaments appear clumped, branched, and anastomosed; morphologically, they resemble the DNA of bacterial nuclei. Mitochondria are absent or degenerate in mature sperm of Cancer crabs, but the centrioles persist in the nucleoplasm at the base of the acrosome.

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Induction of pinocytosis in rat hepatocytes by partial hepatectomy.

The Journal of Cell Biology ◽

10.1083/jcb.72.3.695 ◽

1977 ◽

Vol 72 (3) ◽

pp. 695-706 ◽

Cited By ~ 15

Author(s):

M Mori ◽

A B Novikoff

Keyword(s):

Electron Microscopy ◽

Body Weight ◽

Partial Hepatectomy ◽

Low Dose ◽

Periodic Acid ◽

Light And Electron Microscopy ◽

Rat Hepatocytes ◽

Lysosomal Hydrolases ◽

Periodic Acid Schiff ◽

Perfusion Fixation

Rat hepatocytes, normally not highly pinocytic cells, becomes so after partial hepatectomy when about two-thirds of the liver is removed. Droplets, up to 20 mum in diameter, develop, initially by addition to smaller pinocytic structures and later by fusion with lysosomes. The droplets contain a material with an electron microscope periodicity characteristic of fibrin; they are periodic acid Schiff-positive as is plasma. It is therefore reasonable to consider plasma glycoproteins to be major components of the droplets. The droplets are at all times membrane delimited, an observation possible only after perfusion fixation. The droplets are positive for three lysosomal hydrolases identified cytochemically: acid phosphatase, N-acetyl-beta-glucosaminidase, and beta-glucuronidase. From light and electron microscopy it is evident that these activities are acquired by fusion with lysosomes, mostly autophagic vacuoles and residual bodies both of which become very numerous after partial hepatectomy. Pinocytic structures are seen relatively infrequently in the hepatocytes of normal rats but a great many are present after partial hepatectomy. They are most easily observed if horseradish peroxidase (HRP) is intravenously injected before sacrifice and sections are incubated for HRP cytochemistry. The low dose of HRP employed (10 mg/100 g body weight) does not induce pinocytosis in controls, either untreated rats or rats subjected to laparotomy, including palpation of the liver. However, in partially hepatectomized rats even a much smaller dose of intravenous HRP (3.3 mg/100 g) visualizes the pinocytic structures in hepatocytes (coated vesicles, channels, cuplike bodies, and droplets). Kupffer cells pinocytose much HRP in both control and partially hepatectomized rats.

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The Morphology of Vacuolated Cells in the Liver Following Administration of Vitamin A.

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100072484 ◽

1973 ◽

Vol 31 ◽

pp. 408-409

Author(s):

Odell T. Minick ◽

Hidejiro Yokoo ◽

Fawzia Batti

Keyword(s):

Electron Microscopy ◽

Body Weight ◽

Vitamin A ◽

Light And Electron Microscopy ◽

Liver Cells ◽

Prominent Feature ◽

Vascular Space ◽

Vacuolated Cell ◽

Vacuolated Cells ◽

Vacuolated Cytoplasm

Vacuolated cells in the liver of young rats were studied by light and electron microscopy following the administration of vitamin A (200 units per gram of body weight). Their characteristics were compared with similar cells found in untreated animals.In rats given vitamin A, cells with vacuolated cytoplasm were a prominent feature. These cells were found mostly in a perisinusoidal location, although some appeared to be in between liver cells (Fig. 1). Electron microscopy confirmed their location in Disse's space adjacent to the sinusoid and in recesses between liver cells. Some appeared to be bordering the lumen of the sinusoid, but careful observation usually revealed a tenuous endothelial process separating the vacuolated cell from the vascular space. In appropriate sections, fenestrations in the thin endothelial processes were noted (Fig. 2, arrow).

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Control of Autogenous Vein Grafts Prior to Reimplantation, By Electron Microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010009378x ◽

1972 ◽

Vol 30 ◽

pp. 106-107

Author(s):

John H. L. Watson ◽

John L. Swedo ◽

M. Vrandecic

Keyword(s):

Electron Microscopy ◽

Light And Electron Microscopy ◽

Physiological Saline ◽

Experimental Conditions ◽

Vein Grafts ◽

Arterial Blood ◽

Saphenous Veins ◽

Autogenous Vein ◽

Control Of Parameters ◽

Post Implantation

The ambient temperature and the nature of the storage fluids may well have significant effects upon the post-implantation behavior of venus autografts. A first step in the investigation of such effects is reported here. Experimental conditions have been set which approximate actual operating room procedures. Saphenous veins from dogs have been used as models in the experiments. After removal from the dogs the veins were kept for two hours under four different experimental conditions, viz at either 4°C or 23°C in either physiological saline or whole canine arterial blood. At the end of the two hours they were prepared for light and electron microscopy. Since no obvious changes or damage could be seen in the veins by light microscopy, even with the advantage of tissue specific stains, it was essential that the control of parameters for successful grafts be set by electron microscopy.

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