A Preparation Protocol for Postembedding Immunoelectron Microscopy of Dictyostelium discoideum Cells with Monoclonal Antibodies

Authors

Bruno M. Humbel, University of Utrecht, The Netherlands
Elke Biegelmann, University of Utrecht, The Netherlands

Abstract

On-section-labelling of Dictyostelium discoideum cells poses severe problems in retaining adequate morphology and antigenicity. Monoclonal antibodies are an essential tool in biochemistry and molecular biology because of their specificity and low. background staining. Unfortunately their advantage, the recognition of only one distinct epitope, is often a handicap for immunoelectron microscopy. Resin embedding and steric hindrance of the gold-tagged secondary antibodies further reduce the efficiency in detecting antigens making the localization of less abundant antigens difficult if not impossible. A successful preparation protocol should retain morphology and antigenicity, allow the antibodies easy access to the antigen and use a detection system which visualizes as many of the primary antibodies as possible. Fixation of Dictyostelium cells with buffered formaldehyde and picric acid, cryosectioning and the use of ultra-small gold conjugates enabled us to label most of the antigens under investigation.

Recommended Citation

Humbel, Bruno M. and Biegelmann, Elke (1992) "A Preparation Protocol for Postembedding Immunoelectron Microscopy of Dictyostelium discoideum Cells with Monoclonal Antibodies," Scanning Microscopy: Vol. 6: No. 3, Article 16.
Available at: https://digitalcommons.usu.edu/microscopy/vol6/iss3/16