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.

• Klíčová slova
• Biosensor, Bioseparation, Head and neck cancer, Metallothionein, Nanotechnology,
• 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
• Názvy látek
• baysilon MeSH Prohlížeč
• cadmium telluride MeSH Prohlížeč
• dimethylpolysiloxany MeSH
• metalothionein MeSH
• sloučeniny kadmia MeSH
• telur MeSH
• zlato 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

Centromere is the chromosomal site of kinetochore assembly and microtubule attachment for chromosome segregation. Given its importance, markers that allow specific labeling of centromeric chromatin throughout the cell cycle and across all chromosome types are sought for facilitating various centromere studies. Antibodies against the N-terminal region of CENH3 are commonly used for this purpose, since CENH3 is the near-universal marker of functional centromeres. However, because the N-terminal region of CENH3 is highly variable among plant species, antibodies directed against this region usually function only in a small group of closely related species. As a more versatile alternative, we present here antibodies targeted to the conserved domains of two outer kinetochore proteins, KNL1 and NDC80. Sequence comparison of these domains across more than 350 plant species revealed a high degree of conservation, particularly within a six amino acid motif, FFGPVS in KNL1, suggesting that both antibodies would function in a wide range of plant species. This assumption was confirmed by immunolabeling experiments in angiosperm (monocot and dicot) and gymnosperm species, including those with mono-, holo-, and meta-polycentric chromosomes. In addition to centromere labeling on condensed chromosomes during cell division, both antibodies detected the corresponding regions in the interphase nuclei of most species tested. These results demonstrated that KNL1 and NDC80 are better suited for immunolabeling centromeres than CENH3, because antibodies against these proteins offer incomparably greater versatility across different plant species which is particularly convenient for studying the organization and function of the centromere in non-model species.

• Klíčová slova
• CENH3, Centromere, KNL1, NDC80, immunolabeling, kinetochore,
• MeSH
• centromera * MeSH
• chromatin MeSH
• kinetochory * MeSH
• rostlinné proteiny * genetika   MeSH
• segregace chromozomů MeSH
• sekvence aminokyselin MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chromatin MeSH
• rostlinné proteiny * MeSH

Impaired calcium homeostasis and altered expression of Ca2+-binding proteins are associated with cardiomyopathies, myocardial hypertrophy, infarction or ischemia. S100A1 protein with its modulatory effect on different target proteins has been proposed as one of potential candidates which could participate in these pathological processes. The exact localization of S100A1 in human heart cells on the ultrastructural level accompanied with biochemical determination of its target proteins may help clarify the role of S100A1 in heart muscle. In the present study the distribution of the S100A1 protein using postembedding (Lowicryl K4M) immunocytochemical method in human heart muscle has been determined quantitatively, relating number of antigen sites to the unit area of a respective structural component. S100A1 antigen sites have been detected in elements of sarcoplasmic reticulum (SR), in myofibrils at all levels of sarcomere and in mitochondria, the density of immunolabeling at Z-lines being about 3 times and at SR more than 5 times higher than immunolabeling of remaining structural components. The presence of the S100A1 in SR and myofibrils may be related to the known target proteins for S100A1 at these sites.

• MeSH
• dospělí MeSH
• imunoelektronová mikroskopie MeSH
• imunohistochemie MeSH
• lidé MeSH
• myofibrily chemie   ultrastruktura   MeSH
• myokard chemie   ultrastruktura   MeSH
• proteiny S100 MeSH
• proteiny vázající vápník analýza   MeSH
• sarkomery chemie   ultrastruktura   MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteiny S100 MeSH
• proteiny vázající vápník MeSH
• S100A1 protein MeSH Prohlížeč

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

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 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
• Názvy látek
• akrylové pryskyřice MeSH
• LR white MeSH Prohlížeč

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
• Názvy látek
• aktiny MeSH
• myosin typu I MeSH
• ribozomální DNA MeSH
• RNA ribozomální MeSH
• RNA-polymerasa I MeSH
• transcription factor UBF MeSH Prohlížeč
• transkripční iniciační komplex Pol1 - proteiny MeSH

Methods for identification of apoptotic (AP) cells in tissue sections include light and electron microscopy and immunohistochemical (IHC) detection of apoptotic antigens. Bcl-2 at many tumors inversely correlates with AP and is an indirect marker of AP index. Transglutaminase is often expressed in nonapoptotic cells, and thus represents a non specific marker of AP. Likewise, expression of FAS does not necessarily represent a transformation into AP. IHC detection of caspases does not distinguish between active and nonactive forms of the proteases. Immunolabeling of biotin-conjugated Annexin V is used for the identification of phosphatidylserine residues exposed on the surface of AP cells. Annexin V immuno-gold labeling by means of electron microscopy will allow a more refined description of the morphological events occurring during apoptosis. TUNEL, ISEL and ISNTA methods detect DNA breaks. The rate of AP detected by TUNEL is about 20% higher then by apoptotic figure counting. DNA strand breaks can also occur during DNA repair, electrocoagulation, autolysis, fixation and paraffin embedding. With Apostain, DNA is selectively denaturated by heating with formamide and stained by monoclonal antibody specific to single-strand DNA. It specifically stains condensed chromatin of apoptotic cells. M30 IHC uses a monoclonal antibody binding to the product resulting from cleavage of cytokeratin 18 by activated caspases. M30 is negative in necrotic cells and in progressively degraded cells (AP bodies). In contrast to some pilot studies, we have not reached sufficient sensitivity and specificity of IHC detection with M30 (Roche) in breast carcinomas.

• MeSH
• apoptóza * MeSH
• biopsie MeSH
• imunohistochemie MeSH
• lidé MeSH
• nádory chemie   patologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• anglický abstrakt MeSH
• časopisecké články MeSH
• přehledy MeSH

Macroautophagy is often quantified by live imaging of autophagosomes labeled with fluorescently tagged ATG8 protein (FP-ATG8) in Arabidopsis thaliana. The labeled particles are then counted in single focal planes. This approach may lead to inaccurate results as the actual 3D distribution of autophagosomes is not taken into account and appropriate sampling in the Z-direction is not performed. To overcome this issue, we developed a workflow consisting of immunolabeling of autophagosomes with an anti-ATG8 antibody followed by stereological image analysis using the optical disector and the Cavalieri principle. Our protocol specifically recognized autophagosomes in epidermal cells of Arabidopsis root. Since the anti-ATG8 antibody recognizes multiple AtATG8 isoforms, we were able to detect a higher number of immunolabeled autophagosomes than with the FP-AtATG8e marker, that most probably does not recognize all autophagosomes in a cell. The number of autophagosomes per tissue volume positively correlated with the intensity of autophagy induction. Compared with the quantification of autophagosomes in maximum intensity projections, stereological methods were able to detect the autophagosomes present in a given volume with higher accuracy. Our novel workflow provides a powerful toolkit for unbiased and reproducible quantification of autophagosomes and offers a convenient alternative to the standard of live imaging with FP-ATG8 markers.

• Klíčová slova
• ATG8, Cavalieri principle, autophagosome, autophagy, image analysis, immunofluorescence, immunolabeling, microscopy, optical disector, stereology,
• MeSH
• Arabidopsis * metabolismus   MeSH
• autofagie MeSH
• autofagozomy * metabolismus   MeSH
• kořeny rostlin * metabolismus   MeSH
• proteiny huseníčku metabolismus   MeSH
• průběh práce MeSH
• rodina proteinů Atg8 metabolismus   genetika   MeSH
• zobrazování trojrozměrné metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• proteiny huseníčku MeSH
• rodina proteinů Atg8 MeSH

Simultaneous detection of biological molecules by means of indirect immunolabeling provides valuable information about their localization in cellular compartments and their possible interactions in macromolecular complexes. While fluorescent microscopy allows for simultaneous detection of multiple antigens, the sensitive electron microscopy immunodetection is limited to only two antigens. In order to overcome this limitation, we prepared a set of novel, shape-coded metal nanoparticles readily discernible in transmission electron microscopy which can be conjugated to antibodies or other bioreactive molecules. With the use of novel nanoparticles, various combinations with commercial gold nanoparticles can be made to obtain a set for simultaneous labeling. For the first time in ultrastructural histochemistry, up to five molecular targets can be identified simultaneously. We demonstrate the usefulness of the method by mapping of the localization of nuclear lipid phosphatidylinositol-4,5-bisphosphate together with four other molecules crucial for genome function, which proves its suitability for a wide range of biomedical applications.

• MeSH
• aktiny metabolismus   MeSH
• barvení a značení metody   MeSH
• buněčné jádro MeSH
• elektronová mikroskopie MeSH
• fosfatidylinositol-4,5-difosfát metabolismus   MeSH
• HeLa buňky MeSH
• imunohistochemie metody   MeSH
• jaderné proteiny metabolismus   MeSH
• kovové nanočástice chemie   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nukleofosmin MeSH
• proteiny buněčného cyklu MeSH
• protilátky imunologie   MeSH
• ribonukleoproteiny malé jaderné metabolismus   MeSH
• transportní proteiny metabolismus   MeSH
• zlato chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aktiny MeSH
• fosfatidylinositol-4,5-difosfát MeSH
• jaderné proteiny MeSH
• nukleofosmin MeSH
• proteiny buněčného cyklu MeSH
• protilátky MeSH
• ribonukleoproteiny malé jaderné MeSH
• SMC2 protein, human MeSH Prohlížeč
• transportní proteiny MeSH
• zlato MeSH