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 Cells MeSH
• Histological Techniques methods   MeSH
• Immunohistochemistry * MeSH
• Interphase MeSH
• Cryopreservation * MeSH
• Humans MeSH
• Mitosis * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't 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
• Acrylic Resins MeSH
• Microscopy, Electron MeSH
• HeLa Cells ultrastructure   MeSH
• Histological Techniques methods   MeSH
• Immunohistochemistry methods   MeSH
• Humans MeSH
• Freeze Substitution methods   MeSH
• Preservation, Biological methods   MeSH
• Pressure MeSH
• Tissue Embedding methods   MeSH
• Freezing * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Names of Substances
• Acrylic Resins MeSH
• LR white MeSH Browser

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
• Acrylic Resins * MeSH
• Antigens, Nuclear analysis   MeSH
• Cell Nucleus immunology   ultrastructure   MeSH
• HeLa Cells MeSH
• Immunohistochemistry MeSH
• Cryopreservation methods   MeSH
• Humans MeSH
• Freeze Substitution * MeSH
• Pressure MeSH
• Microscopy, Electron, Transmission MeSH
• Plastic Embedding methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Acrylic Resins * MeSH
• Antigens, Nuclear MeSH
• LR white MeSH Browser