Premium
Aclar discs: a versatile substrate for routine high‐pressure freezing of mammalian cell monolayers
Author(s) -
JIMÉNEZ N.,
HUMBEL B. M.,
VAN DONSELAAR E.,
VERKLEIJ A. J.,
BURGER K. N. J.
Publication year - 2006
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1111/j.1365-2818.2006.01558.x
Subject(s) - monolayer , substrate (aquarium) , biophysics , cell , materials science , electron microscope , cryofixation , microscopy , biomedical engineering , ultrastructure , chemistry , nanotechnology , biology , anatomy , biochemistry , pathology , optics , medicine , ecology , physics
Summary High‐pressure freezing avoids the artefacts induced by conventional chemical fixation, and, in combination with freeze‐substitution and plastic embedding, is a reliable method for the ultrastructural analysis of mammalian cell monolayers. In order to high‐pressure freeze mammalian cell monolayers, cells have to be seeded on a suitable substrate. Unfortunately, electron microscopy analysis is often hampered by poor cell growth, changes in cell morphology induced by the cell substrate or cell loss during processing. We report a method to culture, high‐pressure freeze, freeze‐substitute and plastic embed mammalian cell monolayers. The method is based on the use of Aclar, a copolymer film with properties very similar to those of tissue culture plastic. We show that Aclar discs support the normal growth and morphology of a wide variety of mammalian cell types, and form an ideal starting point for high‐pressure freezing, freeze‐substitution and plastic embedding. We present a complete protocol, which, because of its simplicity and reproducibility, provides a method suitable for the routine analysis of mammalian cell monolayers by electron microscopy and tomography.