bluuebunny/crossref_metadata_2025 huggingface dataset:
Failure of the beige genotype (bg/bg) to alter the morphology of uterine granulated metrial gland cells
Authors: Yamashiro S. , Bast T. , Croy B.A.
Published: June 2020

Granulated metrial gland (GMG) cells are bone marrow-derived cells that differentiate from small, lymphoid-like precursor cells to become large granulated lymphocyte-like cells during murine pregnancy. The cells are localized in the mesometrial triangle and decidua basalis by day 8 of gestation, migrate through the mature placenta and become degenerate in late gestation. The function and lineage of GMG cells are unknown but some experiments suggest that they may be NK cells. Mice of genotype scid/scid have a severe combined immunodeficiency and lack functional B and T lymphocytes. However, both NK cells and GMG cells have been demonstrated in scid/scid mice. To determine whether these two populations are of the same lineage we developed mice of genotype scid/scid,bg/bg, providing a morphological marker, the Chediak-Higashi giant granule phenotype, to NK cells and have asked whether GMG cells share this lymphomyeloid phenotype.

Small pieces of uterine tissues including endo-myometriura from scid/scid and scid/scid.bg/bg at 12th day of gestation were routinely processed for transmission electron microscopy. Ultrastrueturally, large, oval GMG cells were easily recognizable in both scid/scid (Fig. 1) and scid/bg (Fig. 2) uteri due to their cytological features. Nuclei of these cells contained fine chromatin and prominent nucleolus. The cytoplasm was abundant and contained large amounts of free ribosomes and granular endoplasmic reticulum, well developed Golgi complex and prominent membrane-bound granules of variable shapes and sizes (Figs. 1 & 2). The granules were generally oval to spherical and consisted of two zones. The central homogeneous zone, comprising of about 90% of the granule, was less electron opaque than crescent-shaped, outer zone. The crescent-shaped zone contained minute vacuoles and particles (Figs. 1 & 2 insets). The GMG cells exhibited features commonly seen in secretory cells, which were consistent with previous reports. The inclusion characteristic in Chediak-Higashi syndrome was not observed in the GMG cells of the scid/scid.bg/bg (Fig. 2). These observations are suggestive of GMG cells not sharing the NK cell lineage pathway.
Hepatic cytoprotection in treatment of canine heartworm
Authors: Friesen D.L. , Singh A. , Hitt M.E.
Published: June 2020

Thiacetarsamide is an arsenic-containing drug used in the treatment of heartworm in dogs. The effective antihelmintic dose is toxic to the host animal. Acetylcysteine decreases the hepatotoxicity of some compounds by forming a conjugate with toxic metabolites of the compound. The purpose of this study was to evaluate the effectiveness of a cytoprotectant for hepatocytes in dogs treated with therapeutic levels of thiacetarsamide.

Eighteen dogs were divided randomly into two groups. All dogs were given four doses of thiacetarsamide over two days. Nine dogs were given 10% acetylcysteine 15 min prior to each dose of thiacetarsamide. Needle biopsies of the liver were taken from each dog prior to the treatment and again one week post-treatment. The biopsies were fixed in 2% gluraraldehyde in phosphate buffer, pH 7.3, post-fixed in 1% osmium tetroxide, and processed for electron microscopy. Semithin and thin sections of the liver were examined by light and electron microscopy, respectively, for histopathologic and ultrastructural changes. The specimens were coded and the sample treatment was not known to the researchers at the time of observation.
Phagocytosis of pulmonary deposited particles by Type 1 alveolar epithelium
Authors: Sanders C. L. , McDonald K. E. , Adee R. R. , Lauhala K. E.
Published: June 2020

The role of the alveolar epithelium in removal of deposited particles from the alveolar air space has not been well defined. Type II cells, although in close proximity to particles, do not participate in the phagocytosis of particles. How ever, a variety of alveolarly deposited particulates are phagocytized by type I cells. The rapid and efficient phagocytosis of particles in the air space by macrophages minimizes particle entry into more fixed tissues of the lung.

Female, Wistar, young adult rats were given a single intratracheal instillation of either 25 mg iron oxide with a particle size range of 0.3-1.1 micron or 3 mg latex beads with a particle size range of 0.3-0.6 micron, suspended in 1.0 ml 0.9% NaCl solution. Groups of 2-3 rats were killed by halothane overexposure at 5-180 minutes after instillation. The lungs were fixed in situ with McDowellTrumps. Lung tissue was embedded in plastic and stained-with uranyl acetate and lead citrate for electron microscopic examination.
Microscopic study of fetal hypothalamic tissue transplanted into the third ventricle of senescent female rats
Authors: Schneider B.L. , Morley B.J. , Rodriguez-Sierra J.F.
Published: June 2020

As the female rat ages, her ovarian cycles become irregular and she ceases to ovulate. This aging process of reproductive function appears to be partially due to a neurodegenerative process in the hypothalamic areas responsible for the control of the anterior pituitary gland. There has been some success in recovery of function after transplantation of neural tissue in some cases of genetic neural deficits or in deficits due to lesions of the hypothalamus. This experiment was conducted to determine whether the senescent female rat will regain her ability to ovulate after fetal hypothalamic transplants. Here we describe the morphological development of the hypothalamic transplants.

Basal hypothalamic or cortical (control) tissue from fetal rats at 17E were transplanted into senescent Sprague-Dawley female rats demonstrating the cessation of ovulation. Fetal tissue was mechanically dissociated and placed in artificial CSF. Ten μI aliquots of the transplant preparation were infused into the third ventricle of 6 experimental and 6 control animals with a 26 ga Hamilton syringe at a rate of 1 μl/min. The needle remained in place for 5 additional min before it was removed. Animals were monitored for vaginal cyclicity for 30 days. Cyclicity was partially restored in animals transplanted with hypothalamic but not cortical tissue.
Light and electron microscopic demonstration by the PATS reaction of peripheral nerve regeneration
Authors: Giammara B. , Anderson E. , Yates P. , Hanker J.
Published: June 2020

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).
Site of hemocyanin synthesis in the terrestrial isopod, Armadillidiom vulgare
Authors: Faso L. , Rappa E. , Vernon G. , Witkus R.
Published: June 2020

Although hemocyanin, an oxygen binding protein, is found freely dissolved in the hemolymph of isopods its site of synthesis is still unknown.

Circulating blood cel Is such as granular hemocytes have been implicated in hemocyanin synthesis in a number of arthropods including Astacus astacus and Homarus vulgaris. Circulating blood cells of Armadillidium vulgare were examined using a transmission electron microscope (TEM) for evidence of hemocyanin synthesis.

For each experiment hemolymph was collected from twenty adult A. vulgare and fixed for 1 hour in 200 uL of 3.5% glutaraldehyde in 0.1M sodium cacodylate buffer pH 7.4 with 0.05% calcium chloride added. Hemolymph was then centrifuged at 3000 rpm in an IEC-DPR-6000 centrifuge for 15 minutes at 15 degrees centigrade. The supernatant was removed, and the resulting pellet was washed with three changes of sodium cacodylate buffer. Postfixation of the pellet was done in 1%
osmium tetroxide for 1 hour.
Mitosis and cytokinesis in higher plant cells: Scanning Electron Microscopic observations of deplasticized sections
Authors: Russell S.D. , Strout G. W. , Hurd D. M.
Published: June 2020

Plant cell division is a fundamental process of plant development that has been studied by a myriad of different techniques providing principally two-dimensional information. By using fixatives that extract major components of the cytoplasmic matrix, the essential elements of mitotic division can be readily visualized in the scanning electron microscope, and stereopairs can be collected to visually reconstruct a three-dimensional image.

Cells of the onion (Allium cepa L.) were selected for this study as being essentially representative of higher plant cells. Roots were grown over distilled water for 3-5 days, tips were fixed 48h using freshly mixed 1% chromic acid, 10% acetic acid and 37% formaldehyde solution (10:7:3) fixative, dehydrated, embedded in low viscosity resin, sectioned at 2-4 μm, and mounted on gelatin-coated microscope slides. Material was deembedded with 0.5% KOH in 100% methanol, absolute acetone, and benzene (1:1:1) for 30 min; this was neutralized in 1% acetic acid in 100% methanol, rinsed in ethanol, critical point dried in CO2, and coated using Au-Pd in a triode sputter coater. Specimens were viewed on a JEOL JSM-880 SEM at 15 kV using a LaB6 e- source with a probe current <10-11 amperes and a working distance of -1 mm. Stereopairs were photographed at 6° tilt.
Preparation of cultured astrocytes for x-ray microanalysis
Authors: Smith N.K.R. , Hunter K.E. , Mobley P. , Felpel L.P.
Published: June 2020

Electron probe energy dispersive x-ray microanalysis (XRMA) offers a powerful tool for the determination of intracellular elemental content of biological tissue. However, preparation of the tissue specimen , particularly excitable central nervous system (CNS) tissue , for XRMA is rather difficult, as dissection of a sample from the intact organism frequently results in artefacts in elemental distribution. To circumvent the problems inherent in thein vivopreparation, we turned to anin vitropreparation of astrocytes grown in tissue culture. However, preparations ofin vitrosamples offer a new and unique set of problems. Generally, cultured cells, growing in monolayer, must be harvested by either mechanical or enzymatic procedures, resulting in variable degrees of damage to the cells and compromised intracel1ular elemental distribution. The ultimate objective is to process and analyze unperturbed cells. With the objective of sparing others from some of the same efforts, we are reporting the considerable difficulties we have encountered in attempting to prepare astrocytes for XRMA.

Tissue cultures of astrocytes from newborn C57 mice or Sprague Dawley rats were prepared and cultured by standard techniques, usually in T25 flasks, except as noted differently on Cytodex beads or on gelatin. After different preparative procedures, all samples were frozen on brass pins in liquid propane, stored in liquid nitrogen, cryosectioned (0.1 μm), freeze dried, and microanalyzed as previously reported.
Enhanced endothelial cell adherence and spreading on basement membrane matrix coated polystyrene thin films suitable for LM, LV-HR-SEM, and HVEM imaging
Authors: Malecki Marek , Goodman Steven L. , Albrecht Ralph M.
Published: June 2020

Carefully controlled conditions of culture are essential to the in vitro establishment of human primary endothelia] cell cultures. In order to facilitate rapid adherence, spreading, and the development of an in vivo like polygonal pattern of growth, the selection of an appropriate substrate is particularly important. Typically polystyrene “tissue culture” grade dishes subsequently coated with basement membrane proteins, BMM, (collagen, fibronectin, and laminin), or other combinations of extracellular matrix proteins provide an adequate substrate. Correlative light microscopic and electron microscopic examination of cultured whole cell preparations requires that for TEM, HVEM, and STEM, the ceils be grown on a thin film. In addition to the usual requirements of strength and stability, as well as compatability with fixation, staining and dehydration procedures, the films need to be as optically strain free as possible to permit video-enhanced interference or phase based LM.

In preliminary studies we found traditional EM substrates such as formvar and collodion, even when coated with various combinations of matrix proteins, not to be ideal substrates (Fig. 1). Endothelial cell adhesion was reduced, spreading was incomplete even after 4‒6 hours, the rate of cell division was slowed, and the time to confluence was prolonged.
Freeze substitution studies on cultured human fibroblasts
Authors: Heath Julian P. , Turner Donna
Published: June 2020

We are using rapid freezing and freeze substitution to study the three dimensional organisation of membrane systems and cytoskeletal filaments in motile fibroblasts. This study has two objectives: first, to provide material for structural and immunocytochemical analysis of membrane-cytoskeletal interactions in cells that have been preserved with minimum artefact (1,2,3) and second, to refine and develop existing rapid freezing and freeze substitution techniques to allow for the study of single cells that have been experimentally manipulated and observed by digital video microscopy before fixation.

The cells used were human lung fibroblasts (IMR90) either growing on Lux Thermanox coverslips or as pelleted suspensions. The cells were slam frozen on a Med-Vac Cryo Press against a liquid nitrogen cooled copper block. Coverslips were trimmed to 2 x 2 mm in size, excess fluid was drained off, and they were placed on top of a 1mm thick gelatin cushion on an aluminium planchette. For cell suspensions, 3 ul was placed on top of the gelatin cushion. Frozen samples were placed in acetone containing 1% osmium tetroxide for 72 hours at 192 K, wanned to 253 K for 4 hours, and then brought to room temperature. The samples were rinsed in acetone and embedded in Spurr’s resin. Thin sections were cut on a RMC6000 ultramicrotome, stained in uranyl acetate and Reynolds' lead citrate and photographed on a Philips EM410 electron microscope at 60 keV.
The influence of pH on the incorporation of lead in Pisolithus tinctorius
Authors: McQuattie C.J. , Crang R.E.
Published: June 2020

It is recognized that environmental lead contamination due to various forms of air pollution may cause serious damage to plant forest tree species. Much of this damage may occur in its action upon soil microorganisms, particularly fungi which form mycorrhizal associations with fine roots of forest trees. Of particular concern is the mobility of lead ions into plant roots from soils which have become acidified through the influence of atmospheric acidic deposition. Both the mobility and uptake may be affected by pH. In order to determine the lead uptake and extent of cellular damage within fungal cells, we have studied the ultrastructure of lead-treated Pisolithus tinctorius, a common ecto- mycorrhizal symbiont of pine species. Furthermore, we have employed energy- dispersive x-ray microanalysis (EDX) to determine the localization of lead within cells at pH levels of 4.5, 3.5, and 2.5.

Agar cultures of P. tinctorius were used to inoculate defined liquid media adjusted to the three pH levels with 1 N HCl and supplemented with 0, 50 and 100 ppm PbCl2. After growth for 10 days, terminal hyphae were harvested, conventionally fixed for transmission electron microscopy (TEM) with glutaraldehyde and osmium tetroxide, dehydrated in a graded ethanol series, and embedded in an Epon 812/Araldite resin mixture. Ultrathin sections were cut for TEM observations, and semithin sections were prepared for EDX. Sections to be analyzed for lead by means of EDX were unstained.
Diagnosis of avian poxvirus in a red-tailed hawk (Buteo jamaicensis) in Wyoming
Authors: Nunamaker C.E. , Williams E.S. , Driscoll M.P.
Published: June 2020

Avian pox is a contagious viral disease and is worldwide in distribution. A wide variety of domestic and wild birds are affected. Pox infections in birds are characterized by the appearance of discrete, proliferative cutaneous nodules in most cases on the unfeathered parts of the body or by lesions in the upper respiratory and oral mucosa.

In 1987 a red-tailed hawk was submitted to the Wyoming State Veterinary Laboratory, Laramie, WY for diagnosis of a proliferative dermatitis of the feet and head. The bird was trapped in the wild in September, 1986 and had small skin lesions at that time. Skin scrapings were ground with a mortar and pestle, diluted with distilled water and centrifuged at 5000 rpm for 20 min. The supernatant was then centrifuged for one hr at 18,000 rpm. The pellet was resuspended in 10 drops of distilled water. One drop of the suspension was added to a mixture of the following ingredients: 20 drops of distilled water, four drops of 4% phosphotungstic acid and one drop of 0.1% BSA. The mixture was nebulized onto collodion coated 300 mesh copper grids. The material on the grids was then examined with a Philips 410 LS electron microscope for the presence of pox-like particles at 60 KV. Brick shaped pox-like virions measuring 260 X 325 nanometers were observed (Fig. 1). Histopathologic studies revealed large eosinophilic intracytoplasmic inclusion bodies typical of avian poxviral infection in the proliferative epithelium.
Use of conventional embedment procedures for post-embedding immunocytochemistry of secretory proteins
Authors: Purdy-Ramos S. I. , Van Niel E. E. , Forbes M. S. , Carey R. M.
Published: June 2020

The methodology of post-embedding immunocytochemistry commonly employs regimens that are more technically difficult than conventional processing and staining of specimens for transmission electron microscopy. Among these regimens are the preparation of frozen thin sections and embedment in hydrophilic acrylic resins such as Lowicryl and LR White . Cryotechnology, however, requires special equipment and exacting preparation of specimens. Use of water-soluble resins likewise demands special care; for example, embedment in LR White involves titrating the dehydration solutions and maintaining oxygen-free curing conditions (usually in gelatin capsules, making selected orientation of specimens quite difficult). It is commonly supposed that conventional treatments such as exposure to osmium tetroxide and uranyl acetate en bloc, with subsequent embedment in epoxy resins, are detrimental to antigenicity.

Uranyl acetate block-staining can actually enhance immunocytocheinical staining of some epitopes . Though osmicated tissues have also been successfully immunostained , it is standard procedure first to treat the thin sections on grid with an oxidizing agent such sodium metaperiodate . We have found that a polyclonal antibody to renin effectively immunostains globular inclusions (granules) in juxtaglomerular (JG) cells of rat kidney. This can be accomplished both in unosmicated tissue embedded in LR White (Fig. 1) or in specimens (either intact kidney or isolated JG cells) that have been osmicated, uranyl acetate block- stained, dehydrated through 100% ethanol, and embedded in Poly/Bed epoxy resin (cured at 60 °C). Thin sections were collected on nickel grids and immunostained with Protein A-gold, without prior oxidation to remove osmium (Fig. 1). There are several distinct advantages of using the latter immunocytochemical procedure. First, tissues can be processed in a routine manner; in addition, specific orientation of specimens can easily be accomplished. Furthermore, curing of the embedment does not have to be carried out at low temperatures or in oxygen-free containers; this is particularly useful when processing cultured cells (Fig. 1).
A TEM and SEM Study of Campylobacter pylori-like organisms in the human stomach
Authors: Burmeister Veronika , Millikin Paul D. , Taraska John J.
Published: June 2020

Since the studies of Warren (1) and Marshall (2) in 1983, Campylobacter pylori has been isolated with increasing frequency from the human stomach. This spiral organism is located primarily on the surface epithelium, and is very common in cases of active chronic gastritis (6). These findings have led to the exciting speculation that some ulcers, at least, may be secondary to infectious disease, and therefore amenable to antimicrobial treatment.

The specimen described here is from an 80-year old Caucasian male who was originally admitted to the hospital for arthritis and diabetic neuropathy. However, during his rather long hospital stay, he developed a duodenal ulcer which subsequently perforated. An antrectomy was performed. By light microscopy, using the hematoxylin and eosin stain, large numbers of typical spiral organisms were found on the gastric surface. They were readily seen at a magnification of 1000 X, and they also stained strongly with the Warthin-Starry stain. Because the morphology of these organisms was typical for Campylobacter pylori, we considered them to be at least a Campylobacter species. Then, by electron microscopy, we tried to learn whether or not the organisms are physically attached to the epithelial cells.
Demonstration of activated macrophages in subgingival plaque and crevicular fluid samples from periodontal diseased sites
Authors: Hanker J. , Burkes E.J. , Greco G. , Scruggs R. , Anderson E. , Giammara B.
Published: June 2020

Macrophages are present in gingival tissues and they increase in gingivitis and periodontitis. In vitro studies have shown that gram-negative bacterial endotoxin increases the bone resorption activity of macrophages. The activation of macrophages in vitro by substances such as lymphokines, complement, group A streptococcal wall substances, histamine and immune complexes which are present in subgingival plaque or periodontal tissues has also been shown. Intense staining of activated macrophages for acid phosphatase and nonspecific esterase has been observed in tissue sections of inflamed gingiva (cf. 4,9). Although macrophages have also been observed in crevicular fluid and subgingival plaques from normal sites, their numbers, enzyme activities or activation have not previously been studied in crevicular fluid and subgingival plaques from diseased sites. All of the following studies were performed on samples obtained by noninvasive procedures and smeared on coverslips. After cytochemical procedures which produced visible and electron opaque stains on replicate coverslips, correlative light and scanning electron microscopy by SEI and BEI modes permitted identification of cell types and some bacterial pathogens. The PATS reaction, a variation of the periodic acid-Schiff(PAS) reaction that employs stepwise condensation with thiocarbohydrazide and silver methenamine to achieve staining and electron opacity, has been found useful by us for positively staining gram-negative bacteria including spirochetes in crevicular fluid and subgingival plaques from periodontal patients. Neutrophils were intensely stained due to their glycogen content. Larger glycogen positive cells, frequently highly vacuolated, were also present; they appeared to be activated macrophages (Figs. 1-3).
Effects of gossypol acetic acid, a male antifertility drug, on the epididymis of DBA/2J mice
Authors: Majumdar S. K. , Wicks D. L.
Published: June 2020

Recent studies have demonstrated that gossypol acetic acid, a phenolic compound isolated from cotton plants, is a promising reversible male contraceptive in rats and in human beings. Since very little information on the light and ultrastructural effects of the compound on the male reproductive organs in mice is available, this study was undertaken to investigate the effects of the agent on epididymal tissue and spermatozoa in DBA/2J mice.

Gossypol acetic acid was purchased from Sigma Chemical Co., MO. The agent, mixed in olive oil, was administered orally to four groups of mice. Each group received either 0 (control) , 10,20 or 40 mg/kg of the agent twice per week for periods up to 96 days. Animals were sacrificed in pairs at 3,6,9 and 12 weeks. Motility, viability and sperm numbers were determined. Epididymis was fixed in 2% glutaraldehyde, post fixed in 1% osmium tetroxide, dehydrated in an acetone series and embedded in Epon 812. Ultrathin sections were stained and examined under the Philips 200 electron microscope.
Effects of maitotoxin on liver cell cultures as a function of time
Authors: Hassan F.
Published: June 2020

Ciguatera intoxication is caused by the ingestion of fish associated with coral reefs in the tropical and subtropical ecosystems. This poisoning results in serious gastrointestinal, cardiovascular and neurological symptomtologies. The source of this toxicity are a number of epiphytic dinoflagellates. The toxin is transmitted up trophic levels in the food chain. Gambierdiscus toxicus was the first dinoflagellate that was linked to the formation of ciguatoxin. Water soluble toxin from G. toxicus referred to as Maitotoxin (MTX) has been partially purified using high pressure liquid chromatography at SIUC. Some reports are available on the physiological and pharmacological actions of MTX, but there is no evidence whether this toxin has any effects on the ultrastructural morphology of liver cells. Preliminary studies on the effect of MTX on liver cells of chick embryo using light microscopy have shown that it causes necrosis and lesions in this organ. The following study was undertaken to determine whether MTX produces any changes in the ultrastructure of liver cel1 cultures of chick embryo when exposed for different time periods using transmission electron microscopy (TEM).

Fertile chicken eggs were incubated at 101°F for ten days . Their 1ivers were removed aseptically and the cells cultured in medium 199 containing 5% calf serum and 1% antibiotic. After the formation of monolayers, the cells were treated with partially purified MTX at a dosage 568 ng/ml of medium for various time intervals. They were then washed with phosphate buffer at pH 7.4, fixed in buffered 2% formaldehyde, glutaraldehyde, rinsed 3x for 1 hr each in the same buffer at room temp. They were then post fixed in 1% OSO4, stained in uranyl acetate, dehydrated in graded ethanol series, infiltrated with ascending Epon : alcohol mixtures until monolayers were in pure Epon in situ. Gold to silver sections were cut using a diamond knife and were stained with uranyl acetate and lead citrate. The sections were then studied by TEM operated at 50KV.
Cysticercus fasciolaris: Fine surface structural changes in the tegument following in vitro low doses of the antihelminthic drug, oltipraz
Authors: Jones Betty Ruth , Taylor Randolph
Published: June 2020

Cysticercus fasciolaris is the larval form of the adult cat tapeworm, Hydatigera taeniaeformis (Taenia taeniaeformis). It is of increasing interest because of the considerable pathological conditions produced in a rat or mouse host in which it is normally found. The larvae encapsulate and develop on the liver of rats and mice and as a result of this infection, a serious cancer- like growth may develop (Noble and Noble, 1974). The surface tegument of cestodes 1ike that of schistosomes is a highly active layer in which many processes occur (Smyth 1969, 1966). It is a metabolically active syncytial, protoplasmic layer formed by cytoplasmic extensions from tegumental cells lying in the subtegumental zone. The tegument has two major components, namely the distal cytoplasmic zone (surface syncytium) and the perinuclear cytoplasmic zone (cytoplasm in the region of tegumental cells). Under the light microscope the surface of the distal cytoplasmic zone appears as a fringe of fine hair-like processes (Smyth, 1969; Jones et al., 1977a, 1977b Lumsden and Specian, 1980). At the electron microscope level, the distal surface is revealed as spine-like processes which morphologically resemble the brush border of many vertebrate and invertebrate cells and have been referred to as microtriches, microvilli, or tegumental projections (Smyth, 1969 Shivers et al., 1986). The tegument functions to: (1 mediate the absorption of nutrients by the worm from the host environment, (2) protect the integrity of the worm against the alien environment of its host, and (3) bear the immunologicaL mechanisms whereby the parasite is able to resist inactivation by antibodies produced against the invader by its host (Lumsden and Specian, 1980) . Though oltipraz has received less attention than the antihelminthic drug, praziquantel, recent clinical and biological tolerance studies have shown that patients administered the drug have shown a high proportion of egg reduction. The mechanism of action of oltipraz is unclear but there are indications that it might inhibit the parasite’s glutathione levels (Cerf et al. 1982; Bella et al., 1983; Lapierre et al. 1983; Rey et al., 1984; Kardaman et al.,1985; Bieder and Jumeau, 1985), The effects of radioactively labeled praziquantel has been reported in experimental cysticercosis (Anrews et al., 1980). However, the effects of oltipraz on the tegument of cestodes has received little attention. Thus, the objective of this preliminary study is to show the in vitro effects of oltipraz on the tegument of c. fasciolaris in relation to dose and time using scanning electron microscopy.
Towards high-resolution SEM of biological objects
Authors: Müller Martin , Hermann René
Published: June 2020

Electron microscopy is currently the only methodology with the inherent potential to observe structures down to molecular dimensions within the context of complex biological systems. Specimen preparation and imaging techniques should be directed towards preservation and imaging of the smallest significant details in order to fully exploit this unique, integrating feature of biological electron microscopy thereby complementing the progress of the techniques used in cell biology, biochemistry and molecular biology. SEM can contribute much to the understanding of events occurring at the surface of membranes of cells and organelles provided that significant structural details, down to dimensions of 2 - 4 nm, are preserved as a function of the physiological state rather than being determined by the applied specimen preparation procedure. Specimen preparation, signal generation and detection and modern instrumentation have to be interactively optimized with a minimum of compromises.
Tannin-ferrocyanide OsO4 method for scanning electron microscopy with use of microwave irradiation
Authors: Takahashi Gen
Published: June 2020

The disadvantages of metal-coating techniques in high resolution SEM are limitation of resolution due to the thickness of coating, metal decoration of the surface ultrastructures, the lack of uniformity and continuity of coating or specimen damage during coating due to heat generation. In order to overcome these disadvantages and to obtain the better ultrastructural preservation after drying of specimens, the conductive staining methods have been devised.˜4

The microwave irradiation(MWI) has recently been used for fixation of biological specimens for light and electron microscopy, immunohistochemistry and for acceleration of the time required for tissue processing and staining.
In the present study, MWI can be successfully applied to each step in the tannin- ferrocyanide-OsO4(TA- FeCN-Os) method for high resolution SEM.

The TA- FeCNsOs method for high resolution SEM
[A] Primary Osmication(Fixation): double fixation with glutaraldehyde-paraformaldehyde and OsO4, TA-FeCN-Os method for TEM,Osmium-DMSO-Osmium, prolonged osmication or osmication after extraction with saponin, glycerol or detergents.
Investigation of the tridimensional ultrastructure of rat glomerular capillary endothelium by high-resolution scanning electron microscopy
Authors: Lea P.J. , Temkin R.J. , Banoub T. , Silverman M. , Hollenberg M.J.
Published: June 2020

The principal applications of scanning electron microscopy (SEM) to renal ultrastructure have been in the study of the surface topography of various kidney cell types and their orientation and distribution in both health and disease. SEM study, however, has been limited in a major way by a lack of resolution sufficient to readily examine in detail and in three dimensions such features as glomerular basement membrane substructure and the structural organization at high magnification of the glomerular, capillary endothelium. Consequently, most of our current information about the 3D ultrastructure of rat kidney glomerulus has been obtained from transmission electron (TEM) micrographs obtained from thin sections cut at various planes followed by computer assisted, serial reconstructions. Recent advances in specimen preparation techniques and scanning electron microscope design have permitted ultrastructural examination of the glomerular capillary wall in three dimensions using high resolution scanning electron microscopy (HRSEM). Specimens in which the cytosol and cytoskeleton have been extracted, but cell membranes nuclear structures and organelles left in place, were studied using a Hitachi SEM with a resolution of approximately 3 nm.
Quantification of gold labeled cell surface antigens by SEM: Current progress and remaining problems
Authors: de Harven Etienne , Soligo Davide , McGroarty Roy , Christensen Hilary , Leung Richard , Ackerley Cameron
Published: June 2020

Taking advantage of the high elemental contrast of particles of colloidal gold observed in the backscattered electron imaging(BEI) mode of the SEM (1,2), the human T lymphocyte was chosen as a model system to study the potential value of immunogold labeling for the quantification of cell surface expressed molecules. The CD3 antigen which is expressed on all human T lymphocytes and is readily identified by the LEU-4 murine monoclonal antibody (Becton Dickinson, Mountain View, CA) followed by a gold conjugated goat anti-mouse Ig polyclonal antibody was chosen as a model target antigen. When quantified by non-EM methods, using radio-iodinated probes or FACS analysis, approximately 30,000 to 50,000 copies of this antigen per cell are enumerated.

The following observations were made while attempting to quantify the same molecule by SEM after specific immunogold labeling:

Imaging in the SE vs BE mode: The numbers of gold markers counted in the secondary electron (SE) imaging mode are considerably lower than those counted on the same cells in the backscattered electron (BE) imaging mode.
The endocytic pathway in animal cells
Authors: Griffiths Gareth
Published: June 2020

Our recent data have provided evidence that the endocytic pathway comprises four structurally and functionally distinct compartments in addition to the clathrin coated vesicles. The most proximal of these compartments, the early endosome, is structurally complex, consists of cisternal, tubular, as well as vesicular domains and is predominantly localized to the cell periphery. We have proposed that the vesicular domains bud off the early endosome to form the spherical endosome (carrier) vesicles that transport material from early to late endosomes, a process that requires microtubules. These endosome vesicles are spherical (0.2-0.4μm diameter) structures whose lumen is packed with thin tubules. The structure of the latter has recently become more evident in negative stain preparations. These vesicles can also be shown to bind microtubules in vitro (work done in collaboration with Jean Gruenberg's group, EMBL). The third compartment, the late endosome or prelysosomal compartment (PLC) is, like the early endosome, complex in three-dimensions.
Dissection of the molecular mechanisms of protein targeting to the peroxisome using immunofluorescence and immunocryoelectron microscopies
Authors: Keller G-A. , Gould S. J. , Subramani S. , Krisans S.
Published: June 2020

Subcellular compartments within eukaryotic cells must each be supplied with unique sets of proteins that must be directed to, and translocated across one or more membranes of the target organelles. This transport is mediated by cis- acting targeting signals present within the imported proteins. The following is a chronological account of a series of experiments designed and carried out in an effort to understand how proteins are targeted to the peroxisomal compartment.

-We demonstrated by immunocryoelectron microscopy that the enzyme luciferase is a peroxisomal enzyme in the firefly lantern.
-We expressed the cDNA encoding firefly luciferase in mammalian cells and demonstrated by immunofluorescence that the enzyme was transported into the peroxisomes of the transfected cells.
-Using deletions, linker insertions, and gene fusion to identify regions of luciferase involved in its transport to the peroxisomes, we demonstrated that luciferase contains a peroxisomal targeting signal (PTS) within its COOH-terminal twelve amino acid.
Smooth endoplasmic reticulum in perfusion and immersion-fixed Leydig cells
Authors: Bernard R.T.F. , Cross R.H.M.
Published: June 2020

Smooth endoplasmic reticulum (SER) is involved in the biosynthesis of steroid hormones, and changes in the organisation and abundance of this organelle are regularly used as indicators of changes in the level of steroidogenesis. SER is typically arranged as a meshwork of anastomosing tubules which, with the transmission electron microscope, appear as a random mixture of cross, oblique and longitudinal sections. Less commonly the SER appears as swollen vesicles and it is generally suggested that this is an artefact caused during immersion fixation or during immersion of poorly-perfused tissue.

During a previous study of the Leydig cells of a seasonally reproducing bat, in which tissue was fixed by immersion, we noted that tubular SER and vesicular SER often occured in adjacent cells and sometimes in the same cell, and that the abundance of the two types of SER changed seasonally. We came to doubt the widelyheld dogma that vesicular SER was an artefact of immersion fixation and set out to test the hypothesis that the method of fixation does not modify the ultrastructure of the SER.
Freeze-fracture observations on changes in the distribution of Filipin-sterol complexes during epididymal maturation and capacitation of boar spermatozoa
Authors: Seki N. , Toyama Y. , Nagano T.
Published: June 2020

It is believed that i ntramembra.nous sterols play an essential role in membrane stability and permeability. To investigate the distribution changes of sterols in sperm membrane during epididymal maturation and capacitation, filipin has been used as a cytochemical probe for the detection for membrane sterols. Using this technique in combination with freeze fracturing, we examined the boar spermatozoa under various physiological conditions.

The spermatozoa were collected from: 1) caput, corpus and cauda epididymides, 2) sperm rich fraction of ejaculates, and 3)the uterus 2hr after natural coition. They were fixed with 2.5% glutaraldehyde in 0.05M cacodylate buffer (pH 7.4), and treated with the filipin solution (final concentration : 0.02.0.05%) for 24hr at 4°C with constant agitation. After the filipin treatment, replicas were made by conventional freeze-fracture technique. The density of filipin-sterol complexes (FSCs) was determined in the E face of the plasma membrane of head regions.
A morphological study of subitaneous and diapause eggs in marine planktonic copepods
Authors: Santella Luigia , Ianora Adrianna , Dale Brian
Published: June 2020

Many neritic, boreal and sub-temperate copepod species survive periods of environmental adversity by producing over wintering dormant eggs. To understand the nature of arrested development in these crustaceans, we recently studied resting and subitaneous eggs in Pontella mediterranea, a large neustonic copepod that occurs seasonally in Mediterranean surface waters. The species produces morphologically different eggs; smooth eggs that hatch with in 2 d at ca 21°C and spiny eggs that do not hatch under similar conditions. Eggs were fixed for EM immediately after deposition in the case of subitaneous and diapause eggs, and 2 wks and 6 mo later in diapause eggs. Females and eggs were fixed at 4°C in a mixture of 2.5% gluteraldehyde and 1% para formaldehyde buffered to pH 7.2 with 0.2. sodium cacodylate and 20% seawater, rinsed in the same solution with 0.55 M sucrose, and post-fixed and dehydrated in an alcohol series according to conventional procedures.
SEM observations on egg-shell of the Japanese tusser, Antheraea yamamai and Chinese tusser,Antheraea pernyi
Authors: Cui Hu , Hong Jian , Oikang Gao , Xiaojiang Wu , Gongyin Ye
Published: June 2020

The egg-shell surface structure of the Japanese tusser and Chinese tusser was observed by means of scanning electron microscopy. There were a lot of similarities between the two egg-shells, but the fine structure may be easily distinguished. As to the Japanese tusser, the petals of the petaloid pattern around micropyle were elongate and raised in the middle (Fig. 1); micropylar tubes numbered 11-13 (Figs. 2 and 3); the wider and thicker bank formed irregularly shaped net-like structure of the egg-shell surface other than in the vicinity of micropyle (Fig. 5); and the thickness of the egg-shell was about 70 μm. In the Chinese tusser the petals were shorter, wider,and even; micropylar tubes numbered 8-9 (Fig. 4); the narrow and low bank formed hexagonal, pentagonal, heptagonal, or octagonal net-like structure; the aeropyle wall was well developed, almost the same in size (Figs. 8 and 9); and the thickness of the egg-shell was about 40 μm.
Effects of Mg2+ on Chromatic Structure in Isolated Chicken Liver Nuclei
Authors: Arai Soichiro , Nakanishi Yuh H.
Published: June 2020

Although many electron microscopic studies on extracted chromatin have provided considerable information on chromatin condensation induced by divalent cations, there is only a little literature available on the effects of divalent cations on chromatin structure in intact nuclei. In the present study, the effects of Mg2+ on chromatin structure in isolated chicken liver nuclei were examined over a wide concentration range of Mg2+ by scanning electron microscopy.

Nuclei were prepared from chicken liver by the method of Chauveau et al. with some modifications. The nuclei were suspended in 25 mM triethanolamine chloride buffer (pH7.4) with 1 mM EDTA or in the buffer with concentrations of MgCl2 varying from 1 to 50 mM. After incubation for 1 min at 0°C, glutaraldehyde was added to 1.8% and the nuclei were fixed for 1 h at 4°C. The fixed nuclei were mixed with 15% gelatin solution warmed at about 40°C, and kept at room temperature until the mixture set. The gelatin containing the nuclei was fixed with 2% glutaraldehyde for 2-4 h, and cut into small blocks. The gelatin blocks were conductive-stained with 2% tannic acid and 2% osmium tetroxide, dehydrated in a graded series of ethanol, and freeze-cracked with a razor blade in liquid nitrogen.
Scanning Electron Microscopy Study of Cerebellar Synaptic Junctions
Authors: Castejón Orlando J.
Published: June 2020

Conventional and high resolution scanning electron microscopy have been applied to trace short cerebellar nerve circuits and to explore the outer and inner surfaces of spine, glomerular and axodendritic synapses. Samples of teleost fishes (Arius spixii and Salmo Trutta) cerebellar cortex were processed according to the slicing technique for conventional scanning electron microscopy (SEM), ethanol cryofracturing technique and freeze-fracture scanning electron microscopy (FFSEM) (Castejón, 1988). Primate (Rhesus monkey) cerebellar cortex was processed for high resolution scanning electron microscopy (Castejón and Apkarian, 1990), according to the protocol of delicate specimen preparation (Peters, 1980). Observations were made in JEOL 100B with (ASID), scanning attachment ISI DS-130 equiped with LaB6 emitter and Hitachi S-900 SEM with a cold cathode field emitter. Micrographs for HRSEM were soft focus printed to reduce instrumental noise (Peters, 1985). A comparison was made of gold-palladium and chromium coating cerebellar samples.

The slicing technique and the cryofracture process exposed the neuronal outer surface revealing hidden surface ensheathed by neuroglial cells.
Scanning Electron Microscope of Supraependymal Neuronal Circuits in the Third Ventricle of Rhesus Monkey Brain
Authors: Srivastava A.K. , Bajpai V.K. , Singh D.R.
Published: June 2020

The supraependymal neurons have been generally demonstrated lying over the nonciliated areas, specially CVOs, on the walls of the third cerebral ventricle of a variety of mammals. However, such neurons have till date been reported on the ciliated ependyma only inguinea pigs (Mitchell and Card, 1978). In the present study, third ventricles of nine adult monkeys of both the sexes were studied by routine scanning electron microscopy after perfusion fixation in 10 per cent neutral buffered formalin, dehydration with graded series of ethanol and amylacetate, critical-point-drying with liquid carbondioxide and sputter-coating with gold.

Systematic scanning of the lateral wall of the third ventricle revealed that the ependymal surface could be divided into oligo-, and profusely ciliated zones. Pleomorphic supraependymal cells were noticed over the ciliated regions of the ventricularwall particularly between the thalamus and the mammillary region dorsal and caudal to the infundibular recess (Fig. 1). These neuron-like cells typically consisted of cell somas of variable configurations together with extensive networks of interconnected neuritic processes. At higher magnifications, the neuronal bodies depicted diverse structural modifications(Fig.2). Processes of variable diameters coursed over the ciliary carpet of the lateral wall of the third ventricle(Fig.3). At times, the processes exhibiting varicosities over the supramammillaryzone(Fig.4)coursed for variabledistances over the ependymal lining. The majority of such neuritic processes appeared to penetrate the underlying ventricular wall.