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Quantitation of gold labelling and antigens in immunolabelled ultrathin sections.

Immunocytochemistry employs immunological techniques to localise molecular components in biological material. The most commonly used approach is to label protein antigens with antibodies whose location can be revealed with a microscopic marker. At the ultrastructural level the technique is known as immunoelectron microscopy. One of the major challenges in immunoelectron microscopy has been to find ways of exposing the antigens in the specimen to labelling reagents. Currently the method of choice is to make ultrathin 50-100 nm sections. These are then incubated with specific antibodies whose location is revealed using second step reagents such as protein-A complexed to colloidal gold. The colloidal gold particles are used because they have sufficient electron density to be distinguished easily from contrasted cellular structures and subsequently counted. This review focuses exclusively on the quantitative aspects of gold labelling of antigens in cellular structures on ultrathin sections. It first addresses the problem of quantifying gold labelling itself and then focuses on methods which use gold labelling for absolute quantitation of antigens. Many of the approaches that are described rely heavily on principles used in stereology, a science that has been revolutionised recently by key breakthroughs (Gundersen et al. 1988 a, b; Cruz-Orive & Weibel, 1990; Mayhew, 1992).