title: Ultrastructural and nuclear antigen preservation after high-pressure freezing/freeze-substitution and low-temperature LR White embedding of HeLa cells.
abstract: 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.
authors.affiliation: Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, v.v.i., Vídenská 1083, 142 20, Prague, Czech Republic.
authors.forename: Vendula
authors.initials: V
authors.lastname: Strádalová
authors.forename: Katarína
authors.initials: K
authors.lastname: Gaplovská-Kyselá
authors.forename: Pavel
authors.initials: P
authors.lastname: Hozák
mesh_terms: D000180:Acrylic Resins; D034961:Antigens, Nuclear / Q000032:analysis*; D002467:Cell Nucleus / Q000276:immunology* / Q000648:ultrastructure*; D015925:Cryopreservation / Q000379:methods*; D017110:Freeze Substitution; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D046529:Microscopy, Electron, Transmission; D016613:Plastic Embedding / Q000379:methods*; D011312:Pressure
chemical_list: D000180:Acrylic Resins; D034961:Antigens, Nuclear; C048707:LR white
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