Post-embedding Staining with Ferritin Labelled Antibodies

Abstract

THE ferritin labelled antibody technique¹ has been widely used for the location of antigens at the cellular and sub-cellular level². There are two possible methods of applying the technique: (a) pre-embedding staining in which antigen is allowed to react with the labelled antibody before the cells are processed and sectioned in preparation for electron microscopy; and (b) post-embedding staining in which labelled antibody is applied to and reacts with the antigen in suitably fixed and embedded thin sections. Almost all published investigations have been carried out using the pre-embedding staining technique. Surface antigens are readily located by this method, but intra-cellular antigens can only be located after treatments such as freezing and thawing and disintegration which enable the labelled antibody to penetrate within the cell. The technical difficulties associated with the post-embedding staining technique are complicated by the non-specific attraction for ferritin of all commonly used embedding media, including butyl methacrylate–ethyl methacrylate co-polymer, polyglycol methacrylate, vestopal and epon³. The phenomenon was attributed by these workers to the non-ionic character of the embedding media, and they attempted to develop a new embedding medium with ionic charges which allowed wetting, thus reducing nonspecific adsorption. Preliminary results with viral antigens in the polyampholyte embedding medium which they developed were very encouraging.