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.

• Klíčová slova
• Envelope, Immunogold, Labeling, Nuclear, Scanning electron microscopy, Xenopus,
• 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
• Názvy látek
• antigeny MeSH
• epoxidové pryskyřice MeSH
• fixativa MeSH
• formaldehyd MeSH
• koloidní zlato MeSH
• komplex proteinů jaderného póru MeSH
• paraform MeSH Prohlížeč
• polymery MeSH
• protilátky MeSH

The ultrastructural localization of various antigens in a cell using antibodies conjugated to gold particles is a powerful instrument in biological research. However, statistical or stereological tools for testing the observed patterns for significant clustering or colocalization are missing. The paper presents a method for the quantitative analysis of single or multiple immunogold labeling patterns using interpoint distances and tests the method using experimental data. The clustering or colocalization of gold particles was detected using various characteristics of the distribution of distances between them. Pair correlation and cross-correlation functions were used for exploratory analysis; second order reduced K (or cross-K) functions were used for testing the statistical significance of observed events. Confidence intervals of function values were estimated by Monte Carlo simulations of the Poisson process for independent particles, and results were visualized in histograms. Furthermore, a suitability of K functions modified by censoring or weighting was tested. The reliability of the method was assessed by evaluating the labeling patterns of nascent DNA and several nuclear proteins with known functions in replication foci of HeLa cells. The results demonstrate that the method is a powerful tool in biological investigations for testing the statistical significance of observed clustering or colocalization patterns in immunogold labeling experiments.

• MeSH
• buněčné jádro chemie   MeSH
• chemické modely MeSH
• DNA-polymerasa I metabolismus   MeSH
• DNA metabolismus   ultrastruktura   MeSH
• elektronová mikroskopie MeSH
• HeLa buňky MeSH
• imunohistochemie statistika a číselné údaje   MeSH
• lidé MeSH
• replikace DNA MeSH
• S fáze MeSH
• shluková analýza MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• validační studie MeSH
• Názvy látek
• DNA-polymerasa I MeSH
• DNA MeSH

The nuclear pore complex (NPC) is a large elaborate structure embedded within the nuclear envelope, and intimately linked to the cytoskeleton, nucleoskeleton, and chromatin. Many different cargoes pass through its central channel and along the membrane at its periphery. The NPC is dismantled and reassembly, fully or partially, every cell cycle. In post-mitotic cells it consists of a combination of hyper-stable and highly dynamic proteins. Because of its size, dynamics, heterogeneity and integration, it is not possible to understand its structure and molecular function by any one, or even several, methods. For decades, and to this day, thin section transmission electron microscopy (TEM) has been a central tool for understanding the NPC, its associations, dynamics and role in transport as it can uniquely answer questions concerning fine structural detail within a cellular context. Using immunogold labeling specific components can also be identified within the ultrastructural context. Model organisms such as Saccharomyces cerevisiae are also central to NPC studies but have not been used extensively in structural work. This is because the cell wall presents difficulties with structural preservation and processing for TEM. In recent years, high-pressure freezing and freeze substitution have overcome these problems, as well as opened up methods to combine immunogold labeling with detailed structural analysis. Other model organisms such as the worm Caenorhabditis elegans and the plant Arabidopsis thaliana have been underused for similar reasons, but with similar solutions, which we present here. There are also many advantages to using these methods, adapted for use in mammalian systems, due to the instant nature of the initial fixation, to capture rapid processes such as nuclear transport, and preservation of dynamic membranes.

• Klíčová slova
• C. elegans, Freeze substitution, High-pressure freezing, Immunogold, Plant, Thin section transmission electron microscopy, Yeast,
• MeSH
• jaderný pór MeSH
• mrazová substituce * metody   MeSH
• Saccharomyces cerevisiae metabolismus   MeSH
• savci MeSH
• sušené kvasnice * MeSH
• transmisní elektronová mikroskopie MeSH
• zmrazování MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

We present a new method of multiple immunolabeling that is suitable for a broad spectrum of biomedical applications. The general concept is to label both sides of the ultrathin section with the thickness of 70-80 nm with different antibodies conjugated to gold nanoparticles and to distinguish the labeled side by advanced imaging methods with high resolution scanning electron microscopy, such as by correlating images acquired at different energies of primary electrons using different signals. From the Clinical Editor: The use of transmission electron microscopy has become an indispensible tool in the detection of cellular proteins. In this short but interesting article, the authors described their new method of labeling and the identification of four different proteins simultaneously, which represents another advance in imaging technique.

• Klíčová slova
• BSE imaging, Gold nanoparticles, High resolution SEM, Multiple immunolabeling, STEM imaging,
• MeSH
• akrylové pryskyřice chemie   MeSH
• barvení a značení metody   MeSH
• imunohistochemie MeSH
• kovové nanočástice chemie   ultrastruktura   MeSH
• mikrotomie metody   MeSH
• reprodukovatelnost výsledků MeSH
• senzitivita a specificita MeSH
• skenovací elektrochemická mikroskopie metody   MeSH
• vylepšení obrazu metody   MeSH
• zlato chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• akrylové pryskyřice MeSH
• zlato 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

The purpose of this study was to compare two electron microscopy embedding media - LR White and Unicryl - with regard to cell morphologyical and immunohistochemical preservation properties for the study of fixation-sensitive nuclear antigens. Human cervical carcinoma (HeLa) cells were fixed with 2% paraformaldehyde and 0.1% glutaraldehyde, and embedded in parallel in the two resins: LR White and Unicryl using; two different polymerization protocols were used for each resin. Preservation of fine nuclear structure was good after LR White and poor after Unicryl embedding. Immunogold labeling of Sm antigen was significantly stronger on LR White sections. Polymerization by UV light resulted in stronger and more specific labeling than heat polymerization. These results show that LR White is advantageous over Unicryl for the study of nuclear antigens requiring delicate aldehyde fixation.

• MeSH
• akrylové pryskyřice * MeSH
• antigeny jaderné chemie   izolace a purifikace   MeSH
• fixace tkání metody   MeSH
• HeLa buňky MeSH
• imunoelektronová mikroskopie MeSH
• imunohistochemie * MeSH
• indikátory a reagencie MeSH
• lidé MeSH
• ultrafialové záření MeSH
• zalévání tkání MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• akrylové pryskyřice * MeSH
• antigeny jaderné MeSH
• indikátory a reagencie MeSH
• LR white MeSH Prohlížeč
• unicryl MeSH Prohlížeč

Labelling of peripheral blood Lymphocytes surface antigens was carried out using the method of colloidal gold, enhanced with silver staining. Instead of PBS the minimal essential medium (MEM) according the Eagle, pH 7.2, was used rinsing of isolated lymphocytes. Visibility of positive reactions on lymphocytes at application of both mentioned media was the same. Positive reaction at demonstration of p24 BLV on cells acquired the from of black cap while the IgG expression was observed in the from of diffuse dispersion of colloidal gold on cells. Differences between the application of individual media were observed in the shape of peripheral lymphocytes in smears. Utilization of Eagle's MEM resulted in more uniform shapes and optically smooth surfaces when viewed under a light microscope.

• MeSH
• antigeny povrchové krev   MeSH
• barvení stříbrem * MeSH
• imunohistochemie * MeSH
• koncentrace vodíkových iontů MeSH
• lymfocyty imunologie   mikrobiologie   MeSH
• mikroskopie MeSH
• monoklonální protilátky * MeSH
• skot MeSH
• virové proteiny analýza   MeSH
• virus bovinní leukemie chemie   MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny povrchové MeSH
• monoklonální protilátky * MeSH
• virové proteiny MeSH

Polycomb group (PcG) proteins of the Polycomb repressive complex 1 (PRC1) are found to be diffusely distributed in nuclei of cells from various species. However they can also be localized in intensely fluorescent foci, whether imaged using GFP fusions to proteins of PRC1 complex, or by conventional immunofluorescence microscopy. Such foci are termed PcG bodies, and are believed to be situated in the nuclear intechromatin compartment. However, an ultrastructural description of the PcG body has not been reported to date. To establish the ultrastructure of PcG bodies in human U-2 OS cells stably expressing recombinant polycomb BMI1-GFP protein, we used correlative light-electron microscopy (CLEM) implemented with high-pressure freezing, cryosubstitution and on-section labeling of BMI1 protein with immunogold. This approach allowed us to clearly identify fluorescent PcG bodies, not as distinct nuclear bodies, but as nuclear domains enriched in separated heterochromatin fascicles. Importantly, high-pressure freezing and cryosubstitution allowed for a high and clear-cut immunogold BMI1 labeling of heterochromatin structures throughout the nucleus. The density of immunogold labeled BMI1 in the heterochromatin fascicles corresponding to fluorescent "PcG bodies" did not differ from the density of labeling of heterochromatin fascicles outside of the "PcG bodies". Accordingly, an appearance of the fluorescent "PcG bodies" seems to reflect a local accumulation of the labeled heterochromatin structures in the investigated cells. The results of this study should allow expansion of the knowledge about the biological relevance of the "PcG bodies" in human cells.

• Klíčová slova
• BMI1 protein, PcG body, Polycomb group proteins, correlative light-electron microscopy, heterochromatin, high-pressure freezing, immunogold labeling,
• MeSH
• elektronová mikroskopie * MeSH
• heterochromatin metabolismus   MeSH
• imunohistochemie MeSH
• kryoprezervace MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• polycomb proteiny MeSH
• represorové proteiny chemie   metabolismus   MeSH
• světlo * MeSH
• tlak MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• heterochromatin MeSH
• polycomb proteiny MeSH
• represorové proteiny 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.

• Klíčová slova
• Freeze substitution, Immunostaining, Lowicryl, Nuclear, Transmission electron microscopy, Transport, Yeast,
• 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
• Názvy látek
• epoxidové pryskyřice MeSH
• fixativa MeSH
• glutaraldehyd MeSH
• komplex proteinů jaderného póru MeSH
• protilátky MeSH
• Saccharomyces cerevisiae - proteiny MeSH

The PsbH protein belongs to a group of small protein subunits of photosystem II (PSII) complex. This protein is predicted to have a single transmembrane helix and it is important for the assembly of the PSII complex as well as for the proper function at the acceptor side of PSII. To identify the location of the PsbH subunit, the PSII complex with His-tagged PsbH protein was isolated from the cyanobacterium Synechocystis sp. PCC 6803 and labeled by Ni(2+)-nitrilo triacetic acid Nanogold. Electron microscopy followed by single particle image analysis identified the location of the labeled His-tagged PsbH protein at the periphery of the dimeric PSII complex. These results indicate that the N terminus of the PsbH protein is located at the stromal surface of the PSII complex and close to the CP47 protein.

• MeSH
• bakteriální proteiny analýza   ultrastruktura   MeSH
• fosfoproteiny analýza   ultrastruktura   MeSH
• fotosystém II - proteinový komplex analýza   ultrastruktura   MeSH
• imunohistochemie MeSH
• kvarterní struktura proteinů MeSH
• kyselina nitrilotrioctová chemie   MeSH
• makromolekulární látky MeSH
• nikl chemie   MeSH
• podjednotky proteinů analýza   MeSH
• Synechocystis chemie   MeSH
• tylakoidy chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• fosfoproteiny MeSH
• fotosystém II - proteinový komplex MeSH
• kyselina nitrilotrioctová MeSH
• makromolekulární látky MeSH
• nikl MeSH
• photosystem II, psbH subunit MeSH Prohlížeč
• podjednotky proteinů MeSH