Cryostat Sectioning; Modification of the Antiroll Plate

A replaceable antiroll plate and holder have been designed for use in the Ames Lab-Tek cryostat which replace the plastic plate supplied with the instrument and insure a flawless, properly aligned plate for maximum efficiency in thin section cutting. A metal plate holder is attached to the existing screw-driven bracket provided with the instrument by the manufacturer. Glass plates made from one half of a 1.5 × 3 inch microscope slide are coated on the leading edge with spray-on Teflon and provided with tape spacers. These plates slip into the holder and can be adjusted for angular inclination and alignment with the cutting edge by movement within the holder or manipulation of the adjustment screw.     

Improved Methods for Making and Using Glass Knives

We have observed over time that the right side of a glass knife is the optimal cutting edge for microtomy if the counterpiece (heel opposite the edge) is controlled within 1 mm. The right cutting edge has been considered the “saw toothed” side and has not been used for ultrathin sectioning. We have observed that the right cutting edge is sharper and more durable than the left. Light and scanning electron microscopy were used to observe the cutting edge, and transmission electron microscopy was used to examine semithin and ultrathin sections of animal and plant tissues cut by the right and left sides of the cutting edge. The results indicate that the cutting edge becomes sharper and more durable from left to right. Both the quality and efficiency of ultrathin sectioning is improved by using the right cutting edge.     

Some New Methods for Affixing Sections to Glass Slides. I. Aqueous Adhesives

As a new aqueous adhesive to affix sections to glass slides, hydrolyzed vinyl-triethoxysilane-either pure, in combination with polyvinyl alcohol or with specially prepared aqueous polyacrylate solutions-was applied. The silane proved to be very effective in enhancing bonding to the glass surface. As a general aqueous adhesive, a solution of 2% polyvinyl alcohol (m.w. 108,000; 99.7% hydrolyzed) with 0.2% hydrolyzed vinyltriethoxysilane is recommended. This stock solution is diluted 1:10 to 1:50 and used directly to float sections onto slides on a warming plate.     

An Instrument for Non-Destructive Measurement of the Pressure Potential (Turgor) of Leaf Cells

An instrument is described which permits the non-destructivemeasurement of the mean pressure potential (turgor)of leaf laminacells. Calibration shows that the instrument gives a voltageoutput which is linearly related to mean pressure potentialof living leaf cells as determined with a pressure chamber.Measurements may be made very quickly in the field or controlledenvironment with a resolution of at least 50 k Pa.     

Glass capillary columns applied to prostaglandins measurement: a useful tool for gas-chromatography mass spectrometry analysis

The difficulties to analyse prostaglandins (PG) by gas-liquid chromatography are mainly due to the lack of sensitivity of the gas-chromatograph itself (higher than 200 ng) and to the poor resolution of the packed columns. Therefore the use of glass capillary columns which has been applied with success for other biological compounds was tempting. We describe a comparison of the preparation of the columns and their use for PG analysis of standards and of human semen. A complete resolution of PG-1 from PG-2 series was achieved. The sensitivity was multiplied 100 fold with a flame ionisation detector when compared to packed columns and was equal to the one obtained with electron capture detectors without the inconveniences of this technique. The successful coupling of glass capillary columns to a mass spectrometer leads to promising results and allows profile studies of primary PG and their metabolites as seen with human semen.     

A Remodeled Tissue Flattener for Use with the International Microtome Cryostat

The antiroll plate is cut from a standard microscope slide, a 2 cm length, to give a 2 × 2.5 cm piece. This is fitted into inside grooves of a movable metal frame which is held by a hinge joint parallel to the back of the microtome knife. A stationary frame, which supports the hinged member, has spring clips welded to its sides for attachment to the knife. Clearance between the antiroll plate and knife is obtained by applying Scotch tape to the edge of the plate that is adjacent to the knife edge. The hinge permits the plate to be swung back and thus clear the knife surface.     

Glass wool filtration of bull cryopreserved semen: a rapid and effective method to obtain a high percentage of functional sperm

Frozen-thawed bull sperm are widely used in assisted reproductive technologies, but cryopreservation negatively affects semen quality. Several sperm selection techniques have been developed to separate motile sperm from non-motile cells. The aim of the present study was to evaluate the effectiveness of the glass wool column filtration to select functional sperm from frozen-thawed bull semen samples. Frozen semen from six Holstein bulls was thawed and filtered through a glass wool column, followed by assessment of routine and functional sperm parameters. In a set of experiments, sperm aliquots were also processed by swim up to compare both selection methods. Samples recovered in the glass wool filtrate had high percentages of viable (94 ± 3%, mean ± SD), progressively motile (89 ± 4%), acrosome-intact (98 ± 1%), and non-capacitated (80 ± 10%) sperm; these values were higher (P < 0.05) than those obtained after performing the swim up procedure. Moreover, the glass wool filtration yielded 67 ± 19% motile cells, in comparison with 18 ± 8% obtained with swim up (P < 0.05), calculated as the concentration of progressively motile cells selected relative to their concentration in the sample before the selection procedure. Glass wool-filtered sperm were able to undergo capacitation-related events, based on the increase in the percentage of cells classified as capacitated by CTC staining (B-pattern) after incubation with heparin (50 ± 5%) in comparison with control conditions with no heparin (17 ± 4%) or heparin + glucose (16 ± 2%; P < 0.05). Moreover, they underwent acrosomal exocytosis in response to pharmacologic (calcium ionophore A23187 and lysophosphatidylcholine) and physiological (follicular fluid) stimuli, and they fertilized in vitro matured cumulus-oocyte complexes and denuded oocytes (two-cell embryos: 72 ± 4% and 52 ± 6%, respectively). We conclude that glass wool filtration is a low-cost, simple, and highly effective procedure to select functionally competent sperm for reproductive technologies in the bull, which may be useful for other domestic and farm animals, as well as for endangered species.     

Chromatography of Human Leukocyte Interferon on Controlled Pore Glass

Human leukocyte interferon (HuIFN-a) was adsorbed on controlled pore glass (CPG) beads in 0.01 M potassium phosphate, pH 7.2. Partial recovery of HuIFN-a from CPG, ranging from 50% to 75%, was obtained with ethylene glycol (6.8 M), ammonium chloride (1.0 M) and Tris HCl (1.0 M). A near complete recovery (90%) was obtained with potassium phosphate (1 M), but there was no selectivity in the elu-tion vis à vis other proteins. An efficient elution of HuIFN-a was accomplished with tetramethyl-, and tetraethylammonium chlorides: the recovery of HuIFN-a was complete at low concentrations (0.25 M to 0.50 M) of these salts at pH 8.0 and the elution of interferon was more selective than with other eluants.     

A simple and convenient procedure for the hydrogenation of lipids on the micro- and nanomole scale

A glass tube of a special design has been used as a vessel for the hydrogenation of lipids under slight excess pressure at 50 degrees C. Methyl [1-(14)C]linolenate was quantitatively hydrogenated to methyl stearate in less than 30 min. High yields were obtained on both the micromole scale (mean and standard deviation observed for quadruplicate analyses was 98.2 +/- 4.8%) and the nanomole scale (94.3 +/- 7.0%). The applicability of the method is demonstrated by radio-gas-liquid chromatographic analyses of nanomole amounts of (14)C-labeled fatty acid methyl esters from photosynthetic tissue analyzed before and after hydrogenation.     

Development of a surface adhesion immunofluorescent technique for the rapid isolation of Listeria monocytogenes and Listeria innocua from meat

The use of a novel surface adhesion technique to isolate Listeria monocytogenes and Listeria innocua from an enrichment meat system was developed. Minced beef samples inoculated with L. monocytogenes (10 cfu g(-1)) were incubated at 30 degrees C for 14-18 h in a suitable enrichment broth. Listeria monocytogenes cells were isolated from the enriched meat sample by surface adhesion onto a polycarbonate membrane which was attached to a glass microscope slide. The Listeria cells on the membrane were subsequently visualized using an immunofluorescent microscopy procedure. The antibody used in this technique reacts with L. monocytogenes and L. innocua. The technique was demonstrated to have a detection level of log10 3.11 cfu ml(-1). There was excellent correlation (r2 = 0.98) between the counts obtained by this surface adhesion immunofluorescent (SAIF) technique and counts obtained using traditional methods, i.e. plate counts on PALCAM. When the regression equation relating the rapid and standard methods was validated using the data from 50 retail beef mince samples, an rsd value of +/- 0.25 was obtained. No false-negative or false-positive results were recorded for L. monocytogenes or L. innocua species using the SAIF technique.     

New possible mechanisms of embolism formation when measuring vulnerability curves by air injection in a pressure sleeve

Since 1988, researchers have exposed stems to positive pressures to displace water in vessels and measure the impact of applied pressure on hydraulic conductivity. The pressure‐sleeve technique has been used in more than 60 publications to measure vulnerability curves (VCs), which are a measure of how water stress impacts the ability of plants to transport water because water stress induces embolism in vessels that blocks water flow. It is thought that the positive pressure in a sleeve required to induce 50% loss of conductivity (PLC), P₅₀, is the same magnitude as the tension that causes 50% PLC, T₅₀, where the tension can be induced by either bench‐top dehydration or by a centrifuge technique. The unifying concept that P₅₀ = T₅₀ and that the entire VC is the same regardless of method is referred to as the air‐seeding hypothesis. In the current study, we performed experiments to further test the air‐seeding hypothesis in pressure sleeves and concluded that an “effervescence” mechanism caused embolism formation under positive pressure. This mechanism explains why VCs measured using positive pressure do not always match VCs obtained by other methods that induce water tension.     

A Simpler and More Economical Method for Storing Glass Knives

A recently published paper (Schoenwolf 1982) suggested the use of modified microscope slide boxes to store glass knives routinely used for ultramicrotomy. Since the microscope slide boxes cost about $10.00, require modification and may damage the fragile cutting edge unless the knife is carefully oriented, Schoenwolf's method appears to be more expensive and cumbersome than the one used routinely in the authors' laboratory.     

A Method for Breaking Glass Knives Slowly

There is a general belief among electron microscopists that the speed of the break in forming the edge of a glass knife is of great importance in determining its quality. Slow breaks seem very desirable (Hayat 1970).     

Large-Plate Method for the Assay of Neomycin in Serum

A large-plate method employing radial diffusion from small paper discs for assaying serum levels of neomycin is described. More than 120 discs placed on a loading plate and loaded with 20 muliters of sample could all be brought into contact with the agar plate at one time. The requirement for elaborate statistical design to compensate for time-dependent bias was thus eliminated. The dose-response curve was linear for a range of at least 0.5 to 2.5 mug/ml. The experimental limits for the actual zone width (distance from the edge of the paper disc to the outer edge of the inhibition zone) for 120 repetitions of the assay of neomycin in one and the same serum, carried out simultaneously on one large plate, were about +/- 6% (95% confidence interval). The 95% confidence interval for the distribution of difference between duplicate zones obtained for the assay of neomycin in 34 different sera, also carried out on one plate, was about +/- 10%. The dose-response lines for a standard and for three unknown sera, when carried out together on one plate, were parallel within the variability (+/- SD one) of the zone width. The large-plate method is considered to be more efficient than the use of the smaller petri dishes. The method is suitable for the assay of penicillin in serum and can most probably be used for the assay of a wide variety of substances for which radial diffusion from paper discs into agar is feasible.     

High speed detection of circulating tumor cells

Epithelial tumor cells circulate in peripheral blood at ultra-low concentrations in cancer patients. We have developed an instrument capable of rapid and accurate detection of rare cells in circulation utilizing fiber-optic array scanning technology (FAST). The FAST cytometer can locate immunofluorescently labeled rare cells on glass substrates at scan rates 500 times faster than conventional automated digital microscopy. These high scan rates are achieved by collecting fluorescent emissions using a fiber bundle with a large (50 mm) field of view. Very high scan rates make possible the ability to detect rare events without the requirement for an enrichment step. The FAST cytometer was used to detect, image and re-image circulating tumor cells in peripheral blood of breast cancer patients. This technology has the potential to serve as a clinically useful point-of-care diagnostic and a prognostic tool for cancer clinicians. The use of a fixed substrate permits the re-identification and re-staining of cells allowing for additional morphologic and biologic information to be obtained from previously collected and identified cells.     

A method of mesa trimming with glass knives for obtaining large series of ultrathin sections

Ultrastructural analysis of tissue based on 3D reconstruction from serial ultrathin sections is one of the most adequate methods in studies of spatial organization of bio-objects. The sample preparation technique for 3D reconstruction includes the two most technically difficult procedures: an obtaining of stable ribbon of serial sections and mounting of this ribbon onto a slot grid coated with a support film. To mount the ribbon, special approaches and technical tools have been proposed and well evaluated. Much attention has also been paid to obtaining a large and stable ribbon, but this attention deals mainly with the choice of epoxy embedding media. The critical condition of obtaining the straight and stable ribbon is the precise parallelism of trailing and leading edges of mesa falling onto the knife cutting edge. The mesa trimming with dry diamond knife for cryoultratomy allows this condition to be maintained. In the present communication, the way of obtaining parallel sides of the mesa has been proposed with the aid of two forms of glass knives.     

Initiating structural studies of Lys49-PLA2 homologues complexed with an anionic detergent, a fatty acid and a natural lipid

Lys49-Phospholipase A2 (Lys49-PLA(2) - EC 3.1.1.4) homologues damage membranes by a Ca2+-independent mechanism which does not involve catalytic activity. Both MjTX-II from Bothrops moojeni and BthTX-I from Bothrops jararacussu are dimeric in solution and in the crystalline states, and a model for the Ca2+-independent membrane damaging mechanism has been suggested in which flexibility at the dimer interface region permits quaternary structural transitions between "open" and "closed" membrane bound dimer conformations which results in the perturbation of membrane phospholipids and disruption of the bilayer structure. With the aim of gaining insights into the structural determinants involved in protein/lipid association, we report here the crystallization and preliminary X-ray analysis of the (i) MjTX-II/SDS complex at a resolution of 2.78A, (ii) MjTX-II/STE complex at a resolution of 1.8 A and (iii) BthTX-I/DMPC complex at 2.72A. These complexes were crystallized by the hanging drop vapour-diffusion technique in (i) HEPES buffer (pH 7.5) 1.8M ammonium sulfate with 2% (w/v) polyethyleneglycol 400, in (ii) 0.6-0.8 M sodium citrate as the precipitant (pH 6.0-6.5) and in (iii) sodium citrate buffer (pH 5.8) and PEG 4000 and 20% isopropanol, respectively. Single crystals of these complexes have been obtained and X-ray diffraction data have been collected at room temperature using a R-AXIS IV imaging plate system and graphite monochromated Cu Kalpha X-ray radiation generated by a Rigaku RU300 rotating anode generator for (i) and (iii) and using using a Synchrotron Radiation Source (Laboratório Nacional de Luz Sincrotron, LNLS, Campinas, Brazil) for (ii).     

Evaluation of Microflow Digital Imaging Particle Analysis for Sub-Visible Particles Formulated with an Opaque Vaccine Adjuvant

Microflow digital imaging (MDI) has become a widely accepted method for assessing sub-visible particles in pharmaceutical formulations however, to date; no data have been presented on the utility of this methodology when formulations include opaque vaccine adjuvants. This study evaluates the ability of MDI to assess sub-visible particles under these conditions. A Fluid Imaging Technologies Inc. FlowCAM^® instrument was used to assess a number of sub-visible particle types in solution with increasing concentrations of AddaVax^™, a nanoscale squalene-based adjuvant. With the objective (10X) used and the limitations of the sensor resolution, the instrument was incapable of distinguishing between sub-visible particles and AddaVax^™ droplets at particle sizes less than 5 μm. The instrument was capable of imaging all particle types assessed (polystyrene beads, borosilicate glass, cellulose, polyethylene protein aggregate mimics, and lysozyme protein aggregates) at sizes greater than 5 μm in concentrations of AddaVax^™ up to 50% (vol:vol). Reduced edge gradients and a decrease in measured particle sizes were noted as adjuvant concentrations increased. No significant changes in particle counts were observed for polystyrene particle standards and lysozyme protein aggregates, however significant reductions in particle counts were observed for borosilicate (80% of original) and cellulose (92% of original) particles. This reduction in particle counts may be due to the opaque adjuvant masking translucent particles present in borosilicate and cellulose samples. Although the results suggest that the utility of MDI for assessing sub-visible particles in high concentrations of adjuvant may be highly dependent on particle morphology, we believe that further investigation of this methodology to assess sub-visible particles in challenging formulations is warranted.     

Glass fibers covered with sol-gel glass as a new support for affinity chromatography columns: a review.

The search for mechanical supports for biochemically active compounds serving as immunochemical sensors has been the goal of many studies. A new compound in the form of gel fiberglass (GFG) membranes was recently developed as an example of such supports. In this review, these membranes were analyzed with respect to their use for cancer detection. The membranes are prepared from glass fibers covered with oxysilane to provide a sol-gel glass matrix. Derivatization of the support eliminates nonspecific adsorption. A thin layer of protein trapped in the gel glass during its preparation is deposited on the surface of a lattice of glass fibers. The major innovation of the membrane is its large area. External agents percolating through such a membrane contact a maximal number of molecules of the compounds trapped in the sol-gel glass. As a result, this membrane is highly effective. Each GFG column is built from a series of 20 to 30 membranes. The preparation of such columns is relatively simple, requiring only several hours. The capacity of GFG columns is high: the total amount of tumor-associated antigens (TAA) isolated by these columns from the blood of colon cancer patients reached 50% of the total protein and amounted to up to 9-12 mg/ml of serum. The main components of the isolated TAA were the soluble p66 and p51 proteins. A determination of their concentration by HPLC can be used for early cancer detection. Thus, the described method allows the easy and highly effective isolation of TAA and can be used for different goals, including cancer diagnosis. GFG supports have great potential for the isolation of various macromolecules utilizing specific ligands.     

蛋白质微阵列生产用琼脂糖修饰玻片制备的条件优化

目的:建立一种以琼脂糖修饰的玻片为载体的蛋白质微阵列制备的优化方法,比较琼脂糖修饰玻片和醛基修饰玻片及氨基修饰玻片对蛋白质固定效率的优劣。方法:将羊IgG固定在载体表面,经过洗涤、封闭,再加入Cy3标记的兔抗羊IgG,孵育,洗涤后用共聚焦激光扫描仪获取图像,检测各点的荧光强度,根据荧光强度确定最佳琼脂糖浓度,最佳NaIO4浓度,最佳固定时间以及封闭时间等实验条件。结果:琼脂糖浓度为1.2％、NaIO4浓度为20mmol／L、固定时间为1h、孵育时间为45min时,蛋白质在载体上的固定效率和反应活性最高。在固定的抗体浓度相同的情况下,琼脂糖修饰玻片荧光强度是醛基修饰玻片的2.6倍,是氨基修饰玻片的9倍。结论:确立了蛋白质微阵列生产用琼脂糖修饰玻片制备的优化条件,用该优化条件制备的琼脂糖玻片更适合用于蛋白质微阵列载体。