• MeSH
• antigeny MeSH
• imunologické techniky MeSH
• mitochondrie MeSH

• MeSH
• imunoelektronová mikroskopie MeSH

• Konspekt
• Lékařské vědy. Lékařství
• NLK Obory
• diagnostika
• NLK Publikační typ
• kolektivní monografie

• MeSH
• imunochemie MeSH
• játra cytologie   MeSH
• kultivované buňky imunologie   ultrastruktura   MeSH
• myši MeSH
• separace buněk MeSH
• techniky in vitro MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH

Currently, metallothioneins (MTs) are extensively investigated as the molecular biomarkers and the significant positive association of the MT amount was observed in tumorous versus healthy tissue of various types of malignant tumors, including head and neck cancer. Thus, we proposed a biosensor with fluorescence detection, comprising paramagnetic nanoparticles (nanomaghemite core with gold nanoparticles containing shell) for the magnetic separation of MT, based on affinity of its sulfhydryl groups toward gold. Biosensor was crafted from PDMS combined with technology of 3D printing and contained reservoir with volume of 50 μL linked to input (sample/detection components and washing/immunobuffer) and output (waste). For the immunolabeling of immobilized MT anti-MT antibodies conjugated to CdTe quantum dots through synthetic heptapeptide were employed. After optimization of fundamental conditions of the immunolabeling (120 min, 20°C, and 1250 rpm) we performed it on a surface of paramagnetic nanoparticles in the biosensor reservoir, with evaluation of fluorescence of quantum dots (λexc 400 nm, and λem 555 nm). The developed biosensor was applied for quantification of MT in cell lines derived from spinocellular carcinoma (cell line 122P-N) and fibroblasts (122P-F) and levels of the biomarker were found to be about 90 nM in tumor cells and 37 nM in fibroblasts. The proposed system is able to work with low volumes (< 100 μL), with low acquisition costs and high portability.

• MeSH
• 3D tisk * MeSH
• biosenzitivní techniky MeSH
• dimethylpolysiloxany chemie   MeSH
• fluorescence MeSH
• kovové nanočástice MeSH
• kvantové tečky MeSH
• lidé MeSH
• magnetismus MeSH
• metalothionein analýza   MeSH
• nádorové buněčné linie MeSH
• nádory patologie   MeSH
• sloučeniny kadmia chemie   MeSH
• telur chemie   MeSH
• zlato chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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

The endoneurial extracellular matrix (ECM) molecules are involved in cell signalling during nervous system development and regeneration. Quantitative differences of immunofluorescence labelling for chondroitin sulfate proteoglycan (CSPG), fibronectin (FN), tenascin-C (TN-C), and thrombospondin (TSP) were evaluated in intact rat dorsal and ventral roots and dorsal and ventral roots 2 and 4 weeks after rhizotomy using image analysis. The distal stumps of spinal roots displayed increased immunolabelling for the molecules with higher immunofluorescence in dorsal than in ventral roots up to 2 weeks from transection. Four weeks after rhizotomy, the immunoreactivity for CSPG, TN-C and TSP decreased in dorsal and increased in ventral root stumps, although a higher level of immunofluorescence for FN remained in both dorsal and ventral root stumps 4 weeks after injury in comparison to 2 weeks after injury. We suggest that the amount of some ECM molecules changed differentially 2 and 4 weeks after rhizotomy to create an appropriate environment in the endoneurium for early and later regrowth of sensory and motor axons. The results presented here are the first report of differences between the endoneurial ECM content of damaged afferent and motor nerve fibers. In addition, the immunohistochemical detection of individual ECM molecules indicated that final extrinsic conditions stimulating the regrowth of regenerating axons probably arise from a balance of both growth-promoting and -inhibiting molecules in the endoneurium.

• MeSH
• chondroitinsulfát proteoglykany analýza   MeSH
• fibronektiny analýza   MeSH
• financování organizované MeSH
• fluorescenční protilátková technika nepřímá MeSH
• imunohistochemie MeSH
• kryoultramikrotomie MeSH
• krysa rodu rattus MeSH
• míšní kořeny chemie   patologie   růst a vývoj   zranění   MeSH
• motorické neurony patologie   MeSH
• neurony aferentní patologie   MeSH
• periferní nervy chemie   patologie   ultrastruktura   MeSH
• počítačové zpracování obrazu metody   MeSH
• potkani Wistar MeSH
• regenerace nervu MeSH
• rizotomie MeSH
• tenascin analýza   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• srovnávací studie 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

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

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.

• 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

Nuclear actin and nuclear myosin I (NMI) are important players in transcription of ribosomal genes. Transcription of rDNA takes place in highly organized intranuclear compartment, the nucleolus. In this study, we characterized the localization of these two proteins within the nucleolus of HeLa cells with high structural resolution by means of electron microscopy and gold-immunolabeling. We demonstrate that both actin and NMI are localized in specific compartments within the nucleolus, and the distribution of NMI is transcription-dependent. Moreover, a pool of NMI is present in the foci containing nascent rRNA transcripts. Actin, in turn, is present both in transcriptionally active and inactive regions of the nucleolus and colocalizes with RNA polymerase I and UBF. Our data support the involvement of actin and NMI in rDNA transcription and point out to other functions of these proteins in the nucleolus, such as rRNA maturation and maintenance of nucleolar architecture.

• MeSH
• aktiny metabolismus   MeSH
• buněčné jadérko metabolismus   MeSH
• genetická transkripce fyziologie   MeSH
• HeLa buňky MeSH
• imunohistochemie MeSH
• lidé MeSH
• myosin typu I metabolismus   MeSH
• ribozomální DNA metabolismus   MeSH
• RNA ribozomální metabolismus   MeSH
• RNA-polymerasa I metabolismus   MeSH
• transkripční iniciační komplex Pol1 - proteiny metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH