Using quantitative evaluation of immuno-gold labeling and antigen content, we evaluated various automated freeze-substitution protocols used in preparation of biological samples for immunoelectron microscopy. Protein extraction from cryoimmobilized cells was identified as a critical point during the freeze-substitution. The loss of antigens (potentially available for subsequent immuno-gold labeling) was not significantly affected by freezing, while the cryosubstitution with an organic solvent caused a significant loss of antigens. While addition of water can improve visibility of some cell structures, it strengthened the negative effect of cryosubstitution on antigen loss by extraction. This was, however, significantly reversed in the presence of 0.5% glutaraldehyde in the substitution medium. Furthermore, we showed that the level of these changes was antigen-dependent. In conclusion, low concentrations of glutaraldehyde can be generally recommended for cryosubstitution rather than the use of pure solvent, but the exact conditions need to be elaborated individually for certain antigens.

• MeSH
• antigeny jaderné metabolismus   MeSH
• glutaraldehyd MeSH
• HeLa buňky MeSH
• imunoelektronová mikroskopie MeSH
• lidé MeSH
• mrazová substituce metody   MeSH
• rozpouštědla MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH

• MeSH
• imunochemie MeSH
• jaderné proteiny MeSH
• myosiny analýza   fyziologie   MeSH
• vazba proteinů MeSH

• MeSH
• aktiny genetika   MeSH
• buněčné jádro MeSH
• genetická transkripce MeSH
• myosiny genetika   MeSH
• ribozomální DNA MeSH
• RNA-polymerasa I MeSH
• Publikační typ
• techniky in vitro MeSH

• MeSH
• akryláty MeSH
• antigeny chemie   izolace a purifikace   MeSH
• buněčné jádro MeSH
• fixace tkání metody   MeSH
• HeLa buňky MeSH
• imunoelektronová mikroskopie MeSH
• imunohistochemie MeSH
• lidé MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• srovnávací studie 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

• MeSH
• aktiny fyziologie   MeSH
• genetická transkripce genetika   MeSH
• jaderné proteiny MeSH
• myosiny fyziologie   MeSH
• RNA-polymerasa I fyziologie   MeSH

• MeSH
• biotin MeSH
• DNA fyziologie   ultrastruktura   MeSH
• imunochemie metody   MeSH
• lidé MeSH
• replikace DNA MeSH
• S fáze imunologie   MeSH
• zlato MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• techniky in vitro MeSH

• MeSH
• amanitiny farmakologie   MeSH
• buněčné jádro fyziologie   účinky léků   ultrastruktura   MeSH
• genetická transkripce účinky záření   MeSH
• lidé MeSH
• RNA-polymerasa II antagonisté a inhibitory   MeSH
• RNA-polymerasa III antagonisté a inhibitory   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata 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
• 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

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