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
• Názvy látek
• antigeny jaderné MeSH
• glutaraldehyd MeSH
• rozpouštědla 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

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

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
• 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
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• akrylové pryskyřice * MeSH
• antigeny jaderné MeSH
• LR white MeSH Prohlížeč