Preparation of cells and tissues for immuno EM.
Transmission electron microscopy (TEM) provides a powerful set of methods to investigate cellular and subcellular structures using thin sections. In this article we summarize some of the different approaches available for researchers interested in using these methods. The essential details involved in specimen preparation for immunolabelling are covered. The best sectioning approach for preserving specimens for structural analysis is Cryo EM of Vitrified Sections (CEMOVIS), a method where still frozen sections are examined in the transmission electron microscope. Because the specimens are kept at low temperature during sectioning and examination, this method is not amenable for immunolabelling, where antibodies are applied to sections at ambient temperature. To combine structural analysis with immunocytochemical analysis of antigens, the approach of freeze-substitution without chemical fixative is the method of choice, at least from a theoretical point of view. In practice, however, the vast majority of electron microscopic (EM) immunocytochemical analyses are carried out using chemically-fixed specimens that have been embedded in specialized resins (such as the Lowicryls) using freeze-substitution or ambient temperature methods. Antibody labelling of thawed cryosections through chemically-fixed specimens (the Tokuyasu method) is also a popular method for preparing cells and tissues for TEM analysis. Here, we provide an overview of all these sectioning methods for EM, focusing mostly on the practical details. Given the space limitation, the fine details necessary to apply these methods have been successfully omitted and will have to be obtained from the technical references we provide.
Webster
Paul
P
Cell Biology and Biophysics Unit, EMBL, Heidelberg, Meyerhofstrasse 69117, Germany.
Schwarz
Heinz
H
Griffiths
Gareth
G
D000818:Animals; D002477:Cells / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D015925:Cryopreservation / Q000379:methods; D016616:Cryoultramicrotomy / Q000379:methods; D017110:Freeze Substitution / Q000379:methods; D005629:Frozen Sections / Q000379:methods; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D046529:Microscopy, Electron, Transmission / Q000379:methods*; D013048:Specimen Handling / Q000379:methods*; D016610:Tissue Embedding / Q000379:methods
D003432:Cross-Linking Reagents