An optimized device for staining electron microscopy grids

Proper staining of grids is critical for transmission electron microscopy (TEM). Staining must be done as quickly as possible using minimal reagents and with consideration for the environment. We developed a new device for efficient staining of multiple TEM grids. We studied reagent evaporation, rinsing volume, flow rate and re-use of uranyl acetate, and provide here a procedure for efficient staining using the new device. Our device permits TEM grids to be stained with less reagent than alternative staining apparatuses; staining requires a total volume of 260 μl for five grids. Reagent evaporation is less than 6% even if used at 37° C. Moreover, our staining apparatus reduces chemical waste and shortens experiment time by staining several grids simultaneously. Our staining device is a compromise between time-consuming single grid processing and expensive commercial devices that consume large amounts of reagents.     

An optimized protocol for the production of high purity maltose

An economical protocol, which is simple, rapid and reproducible for the production of maltose by enzymatic hydrolysis of tapioca starch, has been optimized. The protocol involves liquefaction of 35% (w/w) tapioca starch by bacterial -amylase at 78±2°C to 3 to 5% (w/w) reducing sugars, followed by maximal (85±3% w/w maltose equivalent) saccharification with barley -amylase and pullulanase at 50°C for 24 to 30 h. The post-saccharification recovery protocol comprised decolourization by charcoal, de-dextrinization by denatured spirit precipitation, de-ionization by passage through cation and anion exchangers and dehydration by vacuum drying. A white crystalline maltose powder was obtained with specifications comparable to commercial high purity maltose. The protocol yields at least 60% (w/w) recovery of maltose and is suitable for use by the pharmaceutical industry. The protocol is unique in that it utilizes cheap and easily hydrolysed tapioca starch, leaves no mother liquor, enabling higher recovery of maltose, and allows almost quantitative recovery of limit maltodextrins, a value-added marketable by-product.     

VIS2FIX: a high-speed fixation method for immuno-electron microscopy

Immuno-transmission electron microscopy (TEM) is the technique of choice for high-resolution localization of proteins in fixed specimen. Here we introduce 2 novel methods for the fixation of sections from cryo-immobilized samples that result in excellent ultrastructural preservation. These high-speed fixation techniques, both called VIS2FIX, allow for a reduction in sample preparation time from at least 1 week to only 8 h. The methods were validated in immuno-TEM experiments on THP-1 monocytes, human umbilical vein endothelial cells (HUVECs) and Madin-Darby canine kidney (MDCK-II) cells. The fixation and retention of neutral lipids is demonstrated, offering unique prospects for the application of immuno-TEM in the lipidomics field. Furthermore, the VIS2FIX methods were successfully employed in correlative fluorescence and electron microscopy.     

Localization of cytoskeletal proteins in Pneumocystis carinii by immuno-electron microscopy

Pneumocystis carinii causes serious pulmonary infection in immunosuppressed patients. This study was undertaken to observe the cytoskeletal proteins of P. carinii by immuno-electron microscopy. P. carinii infection was experimentally induced by immunosuppression of Sprague-Dawley rats for seven weeks, and their lungs were used for the observations of this study. The gold particles localized actin, tropomyosin, and tubulin. The actin was irregularly scattered in the cytoplasm of the trophic forms but was much more concentrated in the inner space of the cell wall of the cystic forms called the inner electron-lucent layer. No significant amount of tropomyosin was observed in either trophic forms or cystic forms. The tubulin was distributed along the peripheral cytoplasm and filopodia of both the trophic and cystic forms rather than in the inner side of the cytoplasm. Particularly, in the cystic forms, the amount of tubulin was increased and located mainly in the inner electron-lucent layer of the cell wall where the actin was concentrated as well. The results of this study showed that the cell wall of P. carinii cystic forms is a structure whose inner side is rich in actin and tubulin. The location of the actin and tubulin in P. carinii suggests that the main role of these proteins is an involvement in the protection of cystic forms from the outside environment by maintaining rigidity of the cystic forms.     

Localization of the azoreductase ofClostridium perfringens by immuno-electron microscopy

An antibody againstClostridium perfringens azoreductase was used with protein A (gold-labeled) to locate the site of synthesis of extracellular azoreductase in this bacterium. Electron microscopy of immunogold-stained thin sections ofC. perfringens cells showed an average of 134 gold particles per cell, distributed throughout the cytoplasm and not associated with any organized structures.     

An optimized protocol for hip joint centre determination using the functional method

The functional method identifies the hip joint centre (HJC) as the centre of rotation of the femur relative to the pelvis during an ad hoc movement normally recorded using stereophotogrammetry. This method may be used for the direct determination of subject-specific HJC coordinates or for creating a database from which regression equations may be derived that allow for the prediction of those coordinates. In order to contribute to the optimization of the functional method, the effects of the following factors were investigated: the algorithm used to estimate the HJC coordinates from marker coordinates, the type and amplitude of the movement of the femur relative to the pelvis, marker cluster location and dimensions, and the number of data samples. This was done using a simulation approach which, in turn, was validated using experiments made on a physical analogue of the pelvis and femur system. The algorithms used in the present context were classified and, in some instances, modified in order to optimize both accuracy and computation time, and submitted to a comparative evaluation. The type of movement that allowed for the most accurate results consisted of several flexion-extension/abduction-adduction movements performed on vertical planes of different orientations, followed by a circumduction movement. The accuracy of the HJC estimate improved, with an increasing rate, as a function of the amplitude of these movements. A sharp improvement was found as the number of the photogrammetric data samples used to describe the movement increased up to 500. For optimal performance with the recommended algorithms, markers were best located as far as possible from each other and with their centroid as close as possible to the HJC. By optimizing the analytical and experimental protocol, HJC location error not caused by soft tissue artefacts may be reduced by a factor of ten with a maximal expected value for such error of approximately 1mm.     

Morphology and structure of lipoproteins revealed by an optimized negative-staining protocol of electron microscopy

Plasma lipoprotein levels are predictors of risk for coronary artery disease. Lipoprotein structure-function relationships provide important clues that help identify the role of lipoproteins in cardiovascular disease. The compositional and conformational heterogeneity of lipoproteins are major barriers to the identification of their structures, as discovered using traditional approaches. Although electron microscopy (EM) is an alternative approach, conventional negative staining (NS) produces rouleau artifacts. In a previous study of apolipoprotein (apo)E4-containing reconstituted HDL (rHDL) particles, we optimized the NS method in a way that eliminated rouleaux. Here we report that phosphotungstic acid at high buffer salt concentrations plays a key role in rouleau formation. We also validate our protocol for analyzing the major plasma lipoprotein classes HDL, LDL, IDL, and VLDL, as well as homogeneously prepared apoA-I-containing rHDL. High-contrast EM images revealed morphology and detailed structures of lipoproteins, especially apoA-I-containing rHDL, that are amenable to three-dimensional reconstruction by single-particle analysis and electron tomography.     

An optimized protocol for isolation of soluble proteins from microalgae for two-dimensional gel electrophoresis analysis

Two-dimensional gel electrophoresis (2-DE)is a core proteomic technique to studyprotein expression and function in livingorganisms. Although it has been extensivelyused for investigation of bacterial, yeast,animal and plant tissue cells, there islimited information about the use of 2-DEin microalgal research. In this study, anumber of key chemical reagents, includingacetone, trichloroacetic acid, urea,thiourea, dithiothreitol, and tributylphosphine, were quantitatively evaluatedfor 2-DE of green microalgae, using Haematococcus pluvialis as a model system.The goal was to maximize the number andstaining intensity of protein spots whileminimizing streaking and smearing on thesecond dimensional SDS gel. Compared tonon-frozen immobilized pH gradients (IPG)strips, freezing of the IPG strips at –20 ^°C after isoelectric focusing (IEF)enhanced protein resolubilization andtransfer into the SDS gel, and thusimproved resolution while eliminatingvertical point streaking on the SDS gel. Itwas also confirmed that manipulation ofsample loading capacity is a simple,effective purification strategy forselective investigation of the proteins ofinterest and of varying abundances. Theprotocol was also successfully applied toprofiling protein expression in H.pluvialis under external stressconditions, indicating its potentialusefulness in further proteomics studies ofthis organism and related species.     

An optimized electroporation protocol for transfection of sensitive cell lines using basic laboratory equipment

Using previously reported protocols, electroporation of 21 very sensitive human cell lines showed poor results with high mortality and low transfection efficiency. Therefore, the influence of several electroporation parameters on transfection success was analyzed. The adjustment of the time constant proved to be most important for optimization of transfection results. Time constant was modulated by changing medium resistance via volume or ionic strength, yielding an average transfection efficiency of 25% and mortality rates below 60%.     

An optimized protocol for metabolome analysis in yeast using direct infusion electrospray mass spectrometry

A method for the global analysis of yeast intracellular metabolites, based on electrospray mass spectrometry (ES-MS), has been developed. This has involved the optimization of methods for quenching metabolism in Saccharomyces cerevisiae and extracting the metabolites for analysis by positive-ion electrospray mass spectrometry. The influence of cultivation conditions, sampling, quenching and extraction conditions, concentration step, and storage have all been studied and adapted to allow direct infusion of samples into the mass spectrometer and the acquisition of metabolic profiles with simultaneous detection of more than 25 intracellular metabolites. The method, which can be applied to other micro-organisms and biological systems, may be used for comparative analysis and screening of metabolite profiles of yeast strains and mutants under controlled conditions in order to elucidate gene function via metabolomics. Examples of the application of this analytical strategy to specific yeast strains and single-ORF yeast deletion mutants generated through the EUROFAN programme are presented.     

Localization of functional centers on the prokaryotic ribosome: immuno-electron microscopy approach

Immuno-electron microscopy studies on the morphology of the Escherichia coli ribosome and the topography of its functional centers are summarized. A three-dimensional model of the ribosome with indications of the mRNA-binding site, the peptidyl-transferase center, the EF-Tu and EF-G-binding sites and the tRNA-binding sites in given. Thus, the mutual arrangement of the centers corresponding to the main functional activities of the ribosome during the process of translation is presented.     

Internalization of CD4 molecules in human T-cells demonstrated by immuno-electron microscopy

Summary Internalization of CD4 molecules on human CD4-enriched T-cells was demonstrated by immunocytochemical electron microscopy. CD4⁺ T-cell subclones were obtained from normal human peripheral blood, followed by one-way MLC screening and co-culturing with IL-2. Fixed and non-fixed T-cell samples were indirectly immunolabeled with mouse anti-human CD4 monoclonal antibody and goat anti-mouse IgG conjugated with peroxidase. Unfixed T-cells were immunolabeled at 4° C and then re-incubated for 5–45 min at 37° C. The selected CD4⁺ T-cell subclones showed strong CD4 binding on the cell surface after IL-2 incubation. However, fresh T-cells, monocytes, bone marrow cells and CD8⁺ T-cells all stained negative for CD4. The distribution of CD4 molecules on the fixed cell surface showed a homogeneous pattern. Capping and internalization of CD4-antibody-peroxidase complexes from the cell surfaces were observed follow a pathway of receptor-mediated endocytosis in unfixed T cells. Endocytotic vesicles, vacuoles of diverse sizes and shapes near the cell membrane or deep in the cell center were found to contain CD4 molecules. Negatively stained Golgi saccules were observed up to 45 min after re-incubation. These results suggest that increased CD4 molecules can be induced on the surface of normal human T-cells in vitro. Internalization and accumulation of CD4 molecules occurred in CD4-enriched T-cells with IL-2 pretreatment.     

An optimized protocol for handling and processing fragile acini cultured with the hanging drop technique

The hanging drop three-dimensional culture technique allows cultivation of functional three-dimensional mammary constructs without exogenous extracellular matrix. The fragile acini are, however, difficult to preserve during processing steps for advanced microscopic investigation. We describe adaptations to the protocol for handling of hanging drop cultures to include investigation using confocal, scanning, and electron microscopy, with minimal loss of cell culture components.     

An optimized protocol for the identification of diatoms,flagellated algae and pathogenic protozoa with phylochips

In the past decade, molecular probe‐based methods have proved successful in improving both the efficiency and accuracy of the identification of microorganisms, especially those that are devoid of distinct morphological features. However until recently, these methods had the major drawback of being limited to the identification of only one or just a few species at a time. With the use of DNA microarrays, it is possible to identify large numbers of taxa on a single‐glass slide, the so‐called phylochip. There are numerous microarray protocols in the literature. These protocols share the same principles, but vary in details, e.g. labelling approach or detergent concentration in the washing buffer. In this study, we show that even small variations in hybridization protocols can have a strong impact on the outcome of the microarray hybridization. An optimized protocol for species identification on phylochips is presented. The optimized protocol is the result of a joined effort of three laboratories to develop phylochips for microbial species identification.     

An optimized MALDI MSI protocol for spatial detection of tryptic peptides in fresh frozen prostate tissue

MALDI MS imaging (MSI) is a powerful analytical tool for spatial peptide detection in heterogeneous tissues. Proper sample preparation is crucial to achieve high quality, reproducible measurements. Here we developed an optimized protocol for spatially resolved proteolytic peptide detection with MALDI time-of-flight MSI of fresh frozen prostate tissue sections. The parameters tested included four different tissue washes, four methods of protein denaturation, four methods of trypsin digestion (different trypsin densities, sprayers, and incubation times), and five matrix deposition methods (different sprayers, settings, and matrix concentrations). Evaluation criteria were the number of detected and excluded peaks, percentage of high mass peaks, signal-to-noise ratio, spatial localization, and average intensities of identified peptides, all of which were integrated into a weighted quality evaluation scoring system. Based on these scores, the optimized protocol included an ice-cold EtOH+H₂O wash, a 5 min heating step at 95°C, tryptic digestion incubated for 17h at 37°C and CHCA matrix deposited at a final amount of 1.8 μg/mm². Including a heat-induced protein denaturation step after tissue wash is a new methodological approach that could be useful also for other tissue types. This optimized protocol for spatial peptide detection using MALDI MSI facilitates future biomarker discovery in prostate cancer and may be useful in studies of other tissue types.     

Internalization of CD4 molecules in human T-cells demonstrated by immuno-electron microscopy.

Internalization of CD4 molecules on human CD4-enriched T-cells was demonstrated by immunocytochemical electron microscopy. CD4+ T-cell subclones were obtained from normal human peripheral blood, followed by one-way MLC screening and co-culturing with IL-2. Fixed and non-fixed T-cell samples were indirectly immunolabeled with mouse anti-human CD4 monoclonal antibody and goat anti-mouse IgG conjugated with peroxidase. Unfixed T-cells were immunolabeled at 4 degrees C and then re-incubated for 5-45 min at 37 degrees C. The selected CD4+ T-cell subclones showed strong CD4 binding on the cell surface after IL-2 incubation. However, fresh T-cells, monocytes, bone marrow cells and CD8+ T-cells all stained negative for CD4. The distribution of CD4 molecules on the fixed cell surface showed a homogeneous pattern. Capping and internalization of CD4-antibody-peroxidase complexes from the cell surfaces were observed follow a pathway of receptor-mediated endocytosis in unfixed T cells. Endocytotic vesicles, vacuoles of diverse sizes and shapes near the cell membrane or deep in the cell center were found to contain CD4 molecules. Negatively stained Golgi saccules were observed up to 45 min after re-incubation. These results suggest that increased CD4 molecules can be induced on the surface of normal human T-cells in vitro. Internalization and accumulation of CD4 molecules occurred in CD4-enriched T-cells with IL-2 pretreatment.