Colloidal gold and biotin-avidin conjugates as ultrastructural markers for neural antigens.

Abstract

The use of two new reagents, biotin-avidin conjugates and colloidal gold particles, for ultrastructural immunocytochemistry has been reviewed, and their potential for immunocytochemistry in the central nervous system has been discussed. Although these compounds have been used very little in the C.N.S. in the past, with their high sensitivity, versatility, and potential for simultaneous localization of two antigens with two electron dense markers, these reagents may prove very useful for answering a number of neural questions. Colloidal gold adsorbed to immunoglobulins can be used for light microscopic observation, and owing to its high electron density and ability to readily adsorb to immunoglobulins, protein A, and antigen-carrier complexes, it also serves as an excellent immunomarker at the ultrastructural level. Several procedures have been described for the use of pre- and post-embedding immunostaining with colloidal gold particles. Different size gold particles, adsorbed to different antibodies can be used for pre- and post-embedding staining of different antigens in the same tissue. While at the light microscopic level only the large particles are visible as a pink haze, a fluorescent step can be added to allow easy identification and characterization of neurones labelled with any size colloidal gold, even if the pink colour of the gold is not visible. These same sections can subsequently be studied ultrastructurally. Unlike enzymatic methods, gold particles have less tendency to obscure ultrastructure. Biotin can be easily conjugated to immunoglobulins and, due to its high affinity for avidin (10(15) M-1) which has four biotin binding sites, can be localized with avidin markers, or biotin-avidin-marker complexes. Some procedures such as the avidin-biotin-peroxidase complex are extremely sensitive. Biotin and avidin can be bound to a number of different markers including peroxidase, colloidal gold, and ferritin. Photomicrographs from our hypothalamic work have been used to illustrate the histological results of some of the procedures described.