Comparison of methods of high-pressure freezing and automated freeze-substitution of suspension cells combined with LR White embedding.
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.
Department of Biology of the Cell Nucleus, Institute of Molecular Genetics of the Academy of Sciences of the Czech Republic, Prague, Czech Republic.
Margarita
M
Sobol
Vlada V
VV
Philimonenko
Pavel
P
Hozák
D000180:Acrylic Resins; D017110:Freeze Substitution / Q000379:methods*; D005615:Freezing; D006367:HeLa Cells / Q000648:ultrastructure; D006652:Histological Techniques / Q000379:methods*; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D008854:Microscopy, Electron; D011309:Preservation, Biological / Q000379:methods; D011312:Pressure; D016610:Tissue Embedding / Q000379:methods*
D000180:Acrylic Resins; C048707:LR white