The spatial distribution of antigens in an ultra-thin sectioned sample can be analysed by application of a post-embedding detection system based on IgG antibodies coupled to ferritin (Wagner et al., 1980) or to colloidal gold particles (Faulk and Taylor, 1971; Romanow et al., 1974, 1975; Roth et al., 1978, 1981; Bendayan et al., 1980; Bendayan, 1982; Bendayan and Zollinger, 1983; Acker, 1988; Rohde et al., 1988; Slot et al., 1988). Pre-conditions are that the position of the antigens is not altered during the preparation procedure, that a sufficient preservation of the ultrastructure is retained, that the antigenicity of the components of interest is preserved, and that the controls indicate the specificity of the antibodies and low background labelling. Both polyclonal antisera and monoclonal antibody preparations may be used. Experience tells us that, due to higher efficiency of labelling, monospecific polyclonal antisera are best suited for this kind of approach. A number of variations of the method can be used:

• Protein A-gold complexes are coupled to the antigen-specific IgG antibody.

• The specific (rabbit) antibody bound to the antigen on the surface of the section is marked by a labelling system consisting of a secondary (goat anti-rabbit) IgG antibody coupled to gold without protein A.

• The antigen is labelled using specific rabbit IgG antibody coupled to colloidal gold without protein A.

The sample preparation may be performed by conventional low temperature embedding (using Lowicryl resins) or by freeze-substitution or cryoultramicrotomy.

Was this article helpful?

0 0

Post a comment