title: A method for efficient observation of intracellular membranes of monolayer culture cells by quick-freeze and freeze-fracture electron microscopy.
abstract: We previously developed a method of analyzing the two-dimensional distribution of membrane lipids by combining quick-freezing and freeze-fracture replica labeling (QF-FRL). In principle, this method can be applied to any membrane, but in practice it is not easy to observe cytoplasmic organelles efficiently without ice crystal damage. In this paper, we report a modification of our method that circumvents this problem. In the modified method, cells are cultured on a gold foil scratched with sandpaper and quick-frozen according to a high-pressure freezing method. This technique enables the efficient observation of intracellular structures such as the nucleus, endoplasmic reticulum, Golgi apparatus and mitochondria. Specific labeling of phosphoinositide 4,5-bisphosphate was confirmed in the obtained freeze-fracture replica. The present QF-FRL method is easy to use and should expedite the analysis of intracellular lipid distribution.
authors.affiliation: Department of Anatomy and Molecular Cell Biology, Nagoya University and Graduate School of Medicine, Nagoya, Japan. afujita@agri.kagoshima-u.ac.jp
authors.forename: Akikazu
authors.initials: A
authors.lastname: Fujita
authors.forename: Toyoshi
authors.initials: T
authors.lastname: Fujimoto
authors.forename: Nami
authors.initials: N
authors.lastname: Ozato-Sakurai
authors.forename: Hitomi
authors.initials: H
authors.lastname: Suzuki
mesh_terms: D002460:Cell Line; D005614:Freeze Fracturing / Q000379:methods*; D006801:Humans; D046529:Microscopy, Electron, Transmission / Q000379:methods*; D015388:Organelles / Q000648:ultrastructure*