Video-enhanced technique for detecting neurophysin, enkephalin, and somatostatin immunoreactivity in ultrathin sections of cat median eminence

A freeze-drying technique using epoxy-embedded ultrathin serial sections permits critical comparisons of neuropeptides in small fibers and varicosities of the nervous system by video-enhanced, light microscopic immunofluorescence. The desirability of the method was documented by data showing: retention of radioimmunoassayable somatostatin in freeze-substituted blocks of tissue as compared to its loss in tissue dehydrated in an alcohol series; feasibility of OsO4 vapor fixation of freeze-dried tissue and compatibility with neuropeptide immunocytochemistry, and utility of a silicon-intensified-tube video camera for recording low levels of fluorescence from ultrathin sections. Ultrathin serial sections, 150 nm thick, from the inner zone of freeze-dried median eminence of the cat revealed three populations of axons containing various combinations of neurophysin immunoreactivity and enkephalin immunoreactivity. Some elements contained neurophysin immunoreactivity alone, some contained both neurophysin immunoreactivity and enkephalin immunoreactivity, and a few elements contained enkephalin immunoreactivity alone. The adjacent external zone of the median eminence contained immunoreactivity for all three substances, but the structures in this region were too small to permit demonstration of coexistence in 150 nm thick sections.     

Development and new design of a semiautomatic cryo-ultramicrotome for histopathological transmission electron microscopy on large ultrathin cryosections: Description of a prototype

In a previous publication, we described new devices and results for diagnostic histopathological transmission electron microscopy on large, ultrathin cryosections from several human tumours. As reported, cryo-ultramicrotomy is gaining in significance and use as technical improvements are made. In comparison with epoxy resin procedures, cryo-preparations are more rapid, there is negligible loss of material and thus antigenicity is well preserved. This work contains some newly developed details for a new type of cryo-ultramicrotome, especially adapted for histopathological investigations on large ultrathin cryosections. It is a software-controlled ultramicrotome advance drive system with feedback regulation and various advancement sensors. Some of these developments could also be applied to existing microtome systems.     

X-ray microanalysis of freeze-dried digestive mucus in Anguilla anguilla L: modifications of ion content during the early steps of secretion

Digestive mucus of sea-water adapted eels has been observed and analyzed by the scanning electron microscope (SEM) after rapid freezing at liquid nitrogen temperature followed by freeze-drying. No chemical procedures were used in this technique. This allowed the maintenance of the mucous coating.Preliminary X-ray microanalysis carried out on freeze-fractured and freeze-dried samples of the oesophagus showed a decrease of K⁺ and an increase of Ca²⁺ and Cl⁻ from the basal part of the mucous cell towards its the apical part. This technique has proven to be satisfactory for it prevents translocation and loss of diffusible elements in situ and allows X-ray microanalysis in the SEM.     

Calcium localization in the shell-forming tissue of the freshwater snail,Biomphalaria glabrata: a comparative study of various methods for localizing calcium

Summary The routes calcium might take across the mantle to the shell have been investigated with various electron-microscopical techniques in the freshwater snailBiomphalaria glabrata (Planorbidae, Basommatophora).In chemically-fixed tissue, calcium was precipitated with a tannic acid-antimonate technique in predominantly the intercellular spaces of the outer mantle epithelium and the interstitium below it. Some vacuoles of the outer mantle epithelium and one type of mucus cell in the inner mantle epithelium also contained precipitate. The presence of calcium in the precipitates was proved by electron energy loss spectroscopy combined with electron spectroscopic imaging. Incubation with lead acetate and uranyl acetate revealed binding-sites for calcium in the intercellular spaces of the epithelia interstitium and the mucus cells of the inner mantle epithelium. Precipitates were also seen after all incubations in the calcium spherites of the connective tissue.The concentrations of calcium and other elements were analysed in freeze-dried ultrathin sections of cryofixed mantle tissue by means of energy-dispersive X-ray microanalysis. Only in mitochondria of the musculature could high amounts of calcium and phosphorous be detected.     

Novel technique for internal structure and elemental distribution analyses of granular sludge from reactors for wastewater treatment

A novel technique for internal structure and elemental distribution analyses of granular sludge is presented. Sludge samples were freeze-dried and embedded in epoxy resin to form a module, which were then ground and polished to obtain sequential cross-sections. The cross-sections were analyzed by scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX). SEM observations showed that one granule was formed having several cores with different inorganic minerals, rather than a single core. EDX results indicate that the main elements of the granules are O, Ca, Mg, and P. In addition, the distribution areas of calcium and magnesium in the granule do not coincide.     

X-ray microanalysis of freeze-dried digestive mucus in Anguilla anguilla L: modifications of ion content during the early steps of secretion

Summary— Digestive mucus of sea-water adapted eels has been observed and analyzed by the scanning electron microscope (SEM) after rapid freezing at liquid nitrogen temperature followed by freeze-drying. No chemical procedures were used in this technique. This allowed the maintenance of the mucous coating. Preliminary X-ray microanalysis carried out on freeze-fractured and freeze-dried samples of the oesophagus showed a decrease of K⁺ and an increase of Ca²⁺ and Cl^? from the basal part of the mucous cell towards its the apical part. This technique has proven to be satisfactory for it prevents translocation and loss of diffusible elements in situ and allows X-ray microanalysis in the SEM.     

Determination of mercury in human serum and packed blood cells by neutron activation analysis

A method is described for the determination of mercury in human blood serum and packed blood cells employing neutron activation analysis. Great attention was devoted to the collection and manipulation of the samples. The accuracy and precision of the method were tested by analyzing biological reference materials and by comparing the concentrations measured in a number of serum samples to those obtained by another, independent technique (cold vapor atomic absorption spectrometry) in the same samples. The article reports the levels measured in blood serum and packed blood cells samples from 15 adult volunteers, as well as the figures determined in a “second-generation” biological reference material (freeze-dried human serum), prepared and conditioned at the University of Ghent.     

Brief communication: Choice of washing method of hair samples for trace element analysis in environmental studies.

Determination of inseparable exogenous and endogenous levels of toxic trace elements such as lead in human scalp hair has been used to understand and assess population exposure to such elements. For any such analysis, washing of the hair samples to remove superficial contamination is the foremost requirement. However, the use of such washing methods prior to elemental analysis removes minute quantities of trace elements from the hair strands. This degree of loss is dependent on the washing method employed. In the present study, the element lead has been determined in human scalp hair after three methods of hair washing. It is apparent that the nonionic detergent-acetone method is best suited for securing valid lead analysis results in human scalp hair.     

Ultrastructural localization of calcium, by electron-probe X-ray microanalysis, in the small intestine of suckling rats

The subcellular distribution of calcium has been investigated in samples, from the intestinal mucosa of 10-day rats, prepared for X-ray microanalysis by various techniques designed to minimize the loss of this element. Calcium retention and its threshold of detection was most satisfactory in freeze-dried frozen thin sections. In resin-embedded samples the best retention of calcium was found in specimens fixed in absolute ethanol, embedded without osmication, and sectioned onto glycerol. The results of this investigation indicate the presence of calcium in the supranuclear vacuole of enterocytes in the distal intestine of the neonatal rat. This calcium is probably taken up during the endocytosis of material from the intestinal lumen. The same mechanism may also be important in the uptake of other metals by suckling animals.     

Efavirenz Dissolution Enhancement IV—Antisolvent Nanocrystallization by Sonication,Physical Stability,and Dissolution

Efavirenz is a fundamental drug in the HIV therapy; however, it has a low bioavailability due to low water solubility. Particle nanonization should enhance its dissolution and therefore its bioavailability. Nanocrystallization is a promising technique for preparing drug nanocrystals. A solution containing efavirenz (EFV) and methanol was added to an aqueous solution of particle stabilizers, under sonication. The adequate polymer stabilizer and its concentration and drug load were evaluated. Particle size and zeta potential of suspensions were measured. Nanosuspensions were freeze-dried and the resulting powder was characterized by some techniques, with special attention to dissolution. Particle size and zeta potential analysis showed that HMPC and PVP were the most suitable polymers. All samples prepared with these stabilizers had nanosized particles and proper zeta potential; however, sedimentation and particle growth were detected with Turbiscan?. Time-related destabilization occurred when the lowest polymer concentration of 20% was used. SEM analysis of the dried powder shows film formation for suspensions with 40% of polymer and particle aggregation in samples with less polymer. Dissolution profiles of samples were higher than EFV raw material, although the lower the polymer concentration, the higher the dissolution.     

Proline: A Novel Cryoprotectant for the Freeze Preservation of Cultured Cells of Zea mays L

Proline is an effective cryoprotectant for the storage of cultured cells of Zea mays L. in liquid N₂. Increased freeze tolerance can be achieved by pregrowth for 3 to 4 days in medium containing proline. Cells cryoprotected with proline have an increased recovery potential when compared with cells cryoprotected with dimethylsulfoxide and glycerol. They also show a reduced postthaw viability loss and greater tolerance of a range of postthaw culture conditions. It is suggested that the mechanism of action of proline may be similar to that in its putative role of conferring protection against natural stresses. It may be protecting the cell against solution effects caused by dehydration during freezing. These findings are discussed in relation to other freeze tolerance enhancing treatments.     

Preparation-dependent distribution of intramembranous particles in freeze-fracture replicas of the neuromuscular junction of the locust Schistocerca gregaria

Summary Freeze-fracture replicas of the neuromuscular junction were prepared from untreated retractor unguis muscles of the locust Schistocerca gregaria that were rapidly frozen by contact with a copper block cooled by liquid helium. These replicas were compared with others prepared from tissue following fixation with glutaraldehyde and cryoprotection in glycerol. Freeze-fracture of rapidly frozen tissue produced replicas of high quality with little evidence of tissue damage by ice crystals in the superficial layers. The gross fracturing characteristics of the neuromuscular junction were consistent with replicas from fixed and cryoprotected tissue; all of the membrane specializations were recognisable but with some alterations in infrastructure. In tissue replicas prepared using either method intramembranous particles in the presynaptic membrane were arranged in a bar-like array. The intramembranous particles of this presynaptic bar array of the rapidly frozen material were large and found on the E-face of the cleaved membrane. This contrasts with the P-face distribution of the comparable particles in muscles fixed in glutaraldehyde and cryoprotected in glycerol, in which they are also smaller and more numerous. This difference in partitioning between rapidly frozen, and fixed, cryoprotected nerve terminals is not found at cholinergic synapses and thus may reflect functional differences between the two types of junction.Indentations of the nerve-terminal membrane occur in replicas from rapidly frozen muscle as well as fixed and cryoprotected muscle suggesting they are not fixation or glycerol-induced artifacts. It is suggested from their position and size that these indentations are more likely to be part of a membrane retrieval system than exocytotic figures.This work was supported by an S.E.R.C. project grant to I.R.D.     

Direct embedding in epoxy resin of cells attached to cellophane

Macrophages were collected from mice or rats after subcutaneous implantation of cellophane or grown in Leighton tubes in which the flying coverslip was replaced by a strip of cellophane. The cellophane and attached cells were prepared for electron microscopy, embedded directly in epoxy resin and then sectioned. The cutting qualities of cellophane are adquate for ultrathin sectioning and permit the ultrastructural examination of the attached macrophage monolayer. The technique can be used for other cell types.     

A Technique for Embedding Undecalcified Bone Samples for Detecting Alpha-Emitters Using Vacuum Impregnation with Spurr's Resin

A method has been developed by which large samples of mineralized bone, containing an alpha-emitter, can be embedded in Spurr's resin in a fraction of the time required by conventional methods. Bone samples were freeze-dried or fixed and dried prior to impregnation with Spurr's resin under vacuum. Sections were cut for the preparation of either alpha-track or fission-track autoradiographs using the solid state detector CR-39. This method is applicable to samples containing a mobile form of a radionuclide that may be translocated during the processes of fixation and dehydration of the specimen.     

Freezing of rat macrophages

The effect of freeze-thawing was studied in cryoprotected (10% dimethyl sulfoxide) rat peritoneal macrophages. Recovery after post-thaw washing was about 50%. Phagocytosis of latex particles seemed unhampered by this procedure, whereas the abilty to adhere to glass and the amount of Fc and C₃ receptors were moderately reduced. Low temperature preservation of macrophages might be a useful storage method, as it is for lymphocytes and tissue culture cells.     

The Effect of a LYSE Exporter Overexpression on l-Arginine Production in Corynebacterium crenatum

A targeted approach for direct topical antimicrobial delivery involving the formulation of impregnated freeze-dried wafers prepared from a natural polymer has been assessed to consider potential for treatment of wounded skin. The synthetic cationic antimicrobial peptides (CAPs) NP101 and NP108 were found to have modest in vitro activity against bacterial species commonly associated with wound infections. Minimum inhibitory concentration/minimum bactericidal concentrations against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa were found to be 0.31 mg/ml for NP101 and 0.25–0.5 mg/ml for NP108. Rapid, substantial cytoplasmic potassium loss was induced by NP108 in E. coli, but not the other species. Through scanning electron microscopy, both CAPs were observed to alter cell morphology, prevent normal septation, promote cell aggregation and trigger release or formation of extracellular filaments. Wafers harbouring these agents displayed substantial antibacterial activity when assessed by standard diffusion assay. These data confirm that topical delivery of CAPs, through their incorporation within freeze-dried wafer formulations prepared from natural polymers, represents a potential viable approach for treating skin infection.     

Titanium and iron titanium oxide nanoparticles in antennae of the migratory ant <Emphasis Type="Italic">Pachycondyla marginata</Emphasis>: an alternative magnetic sensor for magnetoreception?

The most accepted hypothesis of magnetoreception for social insects is the ferromagnetic hypothesis which assumes the presence of magnetic material as a sensor coupled to sensitive structures that transmit the geomagnetic field information to the nervous system. As magnetite is the most common magnetic material observed in living beings, it has been suggested as basic constituent of the magnetoreception system. Antennae and head have been pointed as possible magnetosensor organs in social insects as ants, bees and termites. Samples of three antenna joints: head-scape, scape-pedicel and pedicel-third segment joints were embedded in epoxi resin, ultrathin sectioned and analyzed by transmission electron microscopy. Selected area electron diffraction patterns and X-ray energy dispersive spectroscopy were obtained to identify the nanoparticle compound. Besides iron oxides, for the first time, nanoparticles containing titanium have been identified surrounded by tissue in the antennae of ants. Given their dimension and related magnetic characteristics, these nanoparticles are discussed as being part of the magnetosensor system.     

Durability of phenolic-resin-treated oil palm wood against subterranean termites a white-rot fungus

Oil palm wood (OPW) is seen as a strategic alternative wood material, especially in a country with huge oil-palm-planted areas such as Malaysia. The material is low in quality and various techniques have been used to improve its quality. This study was carried out to evaluate the resistance of low-molecular-weight phenol formaldehyde (Lmw-PF) resin treated OPW against subterranean termites and a white-rot fungus. Four sample groups including untreated OPW and treated OPW samples with three different compression levels (0%, 25%, and 50%) were prepared. Five specimens for each sample group were tested for resistance against subterranean termites (Coptotermes curvignathus) and the white-rot fungus Pycnoporous sanguineus, based on ASTM D 3345-74 and ASTM D 1413-99, respectively. Results showed that both treatment and compression level had significant effects on the percentage weight loss and mean decayed surface of the samples. Treated OPW with 50% compression yielded the best performance with the lowest weight loss on both termite and decay tests. Overall, Lmw-PF resin treated OPW with 25–50% compression can be used as an effective method to improve the durability of OPW.     

An approach to postembedding staining of protein (immunoglobulin) antigen embedded in plastic: prerequisites and limitations

A method is described for performing postembedding staining of protein (immunoglobulin) antigen embedded in styrene-methacrylate resin. Fixation of specimens in a combination of 4% paraformaldehyde and 0.2% picric acid and washing in buffer containing 7% sucrose, followed by abrupt dehydration with absolute acetone in the cold preserved the antigenicity, although in a masked form. The masked antigenicity could be reexposed by treatment with nonspecific protease. Staining with fluorescent-, peroxidase-, or ferritin-labeled antibodies on semi- and ultrathin sections resulted in specific localization of the antigen. We applied this technique to the localization of rabbit immunoglobulin in specimens of renal tissue obtained from rats with anti-glomerular basement membrane nephritis; we also localized human IgG in a renal biopsy specimen. The prerequisites for recovery of antigenicity are such that preservation of tissue structure at the light microscopic level is good, but relatively poor at the electron microscopic level.     

Bacterial Inhibitors Formed During the Adventitious Growth of Micro-organisms in Chicken Liver and Pig Kidney

Inhibitory activity (detected by Bacillus cereus var. mycoides) , identical with that encountered in survey samples, was induced in homogenized fresh chicken liver or pig kidney samples by incubating at 30°C. relative potency of each of three active components as detected by t.l.c./bio-autography of extracts of freeze-dried material was ascertained. Three strains of Streptococcus faecalis and a Lactobacillus sp. were responsible for production of the inhibitory activity, which was not produced by any of these organisms in synthetic liquid media.