In this study we present an optimized method of high-pressure freezing and automated freeze-substitution of cultured human cells, followed by LR White embedding, for subsequent immunolabeling. Also, the influence of various conditions of the freeze-substitution procedures such as temperature, duration, and additives in the substitution medium on the preservation of cryo-immobilized cells was analyzed. The recommended approach combines (1) automated freeze-substitution for high reproducibility and minimizing human-derived errors; (2) minimal addition of contrasting and fixing agents; (3) easy-to-use LR White resin for embedment; (4) good preservation of nuclei and nucleoli which are usually the most difficult structures to effectively vitrify and saturate in a resin; and (5) preservation of antigens for sensitive immunogold labeling.

• MeSH
• akrylové pryskyřice diagnostické užití   MeSH
• elektronová mikroskopie MeSH
• HeLa buňky ultrastruktura   MeSH
• histologické techniky metody   MeSH
• imunohistochemie metody   MeSH
• lidé MeSH
• mrazová substituce metody   MeSH
• ochrana biologická metody   MeSH
• tlak MeSH
• zalévání tkání metody   MeSH
• zmrazování MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

A protocol for high-pressure freezing and LR White embedding of mammalian cells suitable for fine ultrastructural studies in combination with immunogold labelling is presented. HeLa S3 cells enclosed in low-temperature gelling agarose were high-pressure frozen, freeze-substituted in acetone, and embedded in LR White at 0 degrees C. The morphology of such cells and the preservation of nuclear antigens were excellent in comparison with chemically fixed cells embedded in the same resin. The immunolabelling signal for different nuclear antigens was 4-to-13 times higher in high-pressure frozen than in chemically fixed cells. We conclude that one can successfully use high-pressure freezing/freeze-substitution and LR White embedding as an alternative of Lowicryl resins.

• MeSH
• akrylové pryskyřice MeSH
• antigeny jaderné analýza   MeSH
• buněčné jádro imunologie   ultrastruktura   MeSH
• financování organizované MeSH
• HeLa buňky MeSH
• imunohistochemie MeSH
• kryoprezervace metody   MeSH
• lidé MeSH
• mrazová substituce MeSH
• tlak MeSH
• transmisní elektronová mikroskopie MeSH
• zalévání tkání plastickou hmotou metody   MeSH
• Check Tag
• lidé MeSH

Low voltage electron microscopes working in transmission mode, like LVEM5 (Delong Instruments, Czech Republic) working at accelerating voltage 5 kV or scanning electron microscope working in transmission mode with accelerating voltage below 1 kV, require ultrathin sections with the thickness below 20 nm. Decreasing of the primary electron energy leads to enhancement of image contrast, which is especially useful in the case of biological samples composed of elements with low atomic numbers. As a result treatments with heavy metals, like post-fixation with osmium tetroxide or ultrathin section staining, can by omitted. The disadvantage is reduced penetration ability of incident electrons influencing the usable thickness of the specimen resulting in the need of ultrathin sections of under 20 nm thickness. In this study we want to answer basic questions concerning the cutting of extremely ultrathin sections: Is it possible routinely and reproducibly to cut extremely thin sections of biological specimens embedded in commonly used resins with contemporary ultramicrotome techniques and under what conditions? Microsc. Res. Tech. 79:512-517, 2016. © 2016 Wiley Periodicals, Inc.

• MeSH
• design vybavení MeSH
• elektronová mikroskopie přístrojové vybavení   metody   MeSH
• epoxidové pryskyřice chemie   MeSH
• mikrotomie metody   MeSH
• myokard ultrastruktura   MeSH
• myši MeSH
• polymery chemie   MeSH
• srdce diagnostické zobrazování   MeSH
• zalévání tkání plastickou hmotou metody   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

• MeSH
• elektronová mikroskopie MeSH
• histologické techniky MeSH

The best available approach of biological sample preparation for transmission electron microscopy currently includes cryoimmobilization by high-pressure freezing (HPF) followed by freeze-substitution (FS). This method has been well established for interphase cells; however, a reliable and easy procedure is still missing for mitotic cells especially because of their fragility and sensitivity to treatments. Here, we present a fast and effective method for HPF/automated FS and LR White embedding of mitotic cells which allows for their controlled and reproducible quality processing. It should be useful in various ultrastructural studies on mitotic cells especially in combination with immunocytochemistry.

• MeSH
• HeLa buňky MeSH
• histologické techniky metody   MeSH
• imunohistochemie MeSH
• interfáze MeSH
• kryoprezervace MeSH
• lidé MeSH
• mitóza MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• Klíčová slova
• protargol,
• MeSH
• elektronová mikroskopie MeSH
• epoxidové pryskyřice MeSH
• glykogen MeSH
• mikroskopie MeSH
• stříbrné proteiny MeSH

Electrodialysis and electrodeionization are separation processes whose performance depends on the quality and properties of ion-exchange membranes. One of the features that largely affects these properties is heterogeneity of the membranes both on the macroscopic and microscopic level. Macroscopic heterogeneity is an intrinsic property of heterogeneous ion-exchange membranes. In these membranes, the functional ion-exchange component is dispersed in a non-conductive binder. The functional component is finely ground ion-exchange resin particles. The understanding of the effect of structure on the heterogeneous membrane properties and behavior is thus of utmost importance since it does not only affect the actual performance but also the cost and therefore competitiveness of the aforementioned separation processes. Here we study the electrokinetic behavior of cation-exchange resin particle systems with well-defined geometrical structure. This approach can be understood as a bottom up approach regarding the membrane preparation. We prepare a structured cation-exchange membrane by using its fundamental component, which is the ion exchange resin. We then perform an experimental study with four different experimental systems in which the number of used cation-exchange particles changes from 1 to 4. These systems are studied by means of basic electrochemical characterization measurements, such as measurement of current-voltage curves and direct optical observation of phenomena that occur at the interface between the ion-exchange system and the adjacent electrolyte. Our work aims at better understanding of the relation between the structure and the membrane properties and of how structure affects electrokinetic behavior of these systems.

• MeSH
• elektřina * MeSH
• elektrochemie MeSH
• iontoměniče chemie   MeSH
• iontová výměna * MeSH
• kationty chemie   MeSH
• koncentrace vodíkových iontů MeSH
• membrány umělé MeSH
• Publikační typ
• časopisecké články MeSH

In this study, we describe an approach that enables a highly specific, effective and fast detection of polyadenylated RNA sequences in situ at the light and electron microscopy levels. The method developed is based on the incorporation of 5-bromo-2'-deoxyuridine into the growing cDNA strand by means of the reverse transcriptase. We have shown that unlike the previously used deoxyuridine tagged with biotin or digoxigenin, 5-bromo-2'-deoxyuridine is 'invisible' in the DNA-DNA duplex but easily detectable in the DNA-RNA duplex. This feature is an important pre-requisite for the correct interpretation of the data obtained, as our results strongly indicate that reverse transcriptase uses DNA breaks as primers efficiently. We have also shown that the replacement of deoxythymidine by 5-bromo-2'-deoxyuridine considerably stabilizes the growing DNA-RNA duplex, thus enabling the one-step detection of polyadenylated RNA in structurally well-preserved cells. The method developed provides a highly specific signal with the signal/noise ratio higher than 130 for permeabilized cells and 25 for conventional acrylic resin sections under the conditions used. When the high pressure freezing technique followed by the freeze substitution is employed for the cell's preparation, the ratio is higher than 80.

• MeSH
• akrylové pryskyřice MeSH
• fluorescenční mikroskopie MeSH
• HeLa buňky MeSH
• hybridizace in situ fluorescenční metody   MeSH
• lidé MeSH
• messenger RNA analýza   chemie   MeSH
• permeabilita buněčné membrány MeSH
• poly A analýza   MeSH
• reverzní transkripce MeSH
• zalévání tkání MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Scanning electron microscopes are useful biological tools that can be used to image the surface of whole organisms, tissues, cells, cellular components, and macromolecules. Processes and structures that exist at surfaces can be imaged in pseudo, or real 3D at magnifications ranging from about 10× to 1,000,000×. Therefore a whole multicellular organism, such as a fly, or a single protein embedded in one of its cell membranes can be visualized. In order to identify that protein at high resolution, or to see and quantify its distribution at lower magnifications, samples can be labeled with antibodies. Any surface that can be exposed can potentially be studied in this way. Presented here is a generic method for immunogold labeling for scanning electron microscopy, using two examples of specimens: isolated nuclear envelopes and the cytoskeleton of mammalian culture cells. Various parameters for sample preparation, fixation, immunogold labeling, drying, metal coating, and imaging are discussed so that the best immunogold scanning electron microscopy results can be obtained from different types of specimens.

• MeSH
• antigeny genetika   metabolismus   MeSH
• barvení a značení metody   MeSH
• buněčná membrána metabolismus   ultrastruktura   MeSH
• cytoskelet metabolismus   ultrastruktura   MeSH
• epoxidové pryskyřice chemie   MeSH
• exprese genu MeSH
• fixace tkání metody   MeSH
• fixativa chemie   MeSH
• formaldehyd chemie   MeSH
• imunohistochemie metody   MeSH
• jaderný obal metabolismus   ultrastruktura   MeSH
• koloidní zlato chemie   MeSH
• komplex proteinů jaderného póru genetika   metabolismus   MeSH
• mikroskopie elektronová rastrovací metody   MeSH
• mikrotomie MeSH
• oocyty metabolismus   ultrastruktura   MeSH
• polymery chemie   MeSH
• protilátky chemie   MeSH
• Xenopus laevis MeSH
• zalévání tkání metody   MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Immunolabeling electron microscopy is a challenging technique with demands for perfect ultrastructural and antigen preservation. High-pressure freezing offers an excellent way to fix cellular structure. However, its use for immunolabeling has remained limited because of the low frequency of labeling due to loss of protein antigenicity or accessibility. Here we present a protocol for immunogold labeling of the yeast Saccharomyces cerevisiae that gives specific and multiple labeling while keeping the finest structural details. We use the protocol to reveal the organization of individual nuclear pore complex proteins and the position of transport factors in the yeast Saccharomyces cerevisiae in relation to actual transport events.

• MeSH
• barvení a značení metody   MeSH
• epoxidové pryskyřice chemie   MeSH
• exprese genu MeSH
• fixace tkání metody   MeSH
• fixativa chemie   MeSH
• glutaraldehyd chemie   MeSH
• imunoelektronová mikroskopie metody   MeSH
• imunohistochemie metody   MeSH
• komplex proteinů jaderného póru genetika   metabolismus   MeSH
• kryoprezervace metody   MeSH
• mikrotomie MeSH
• mrazová substituce metody   MeSH
• protilátky chemie   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae metabolismus   ultrastruktura   MeSH
• zalévání tkání metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH