bluuebunny/crossref_metadata_2025_split huggingface dataset:
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.
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.
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.˜4The 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.
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.
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.
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.