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Abstract

The application of freeze-substitution (FS) and freeze-drying (FD) techniques and the protein A-gold-antibody complex immunocytochemical methods are described. The two tissue-preparation techniques produced excellent ultrastructure and topographical fixation of antigens when compared with conventional tissue-preparation techniques. In the FS preparation, however, occasional extragranular immunolabeling was recognized. This may suggest the leakage of antigens from the secretory granules. The FD procedure was considered the best, since such labeling was almost negligible. The protein A-gold-antibody complexes are easily prepared and label the antigens clearly. If the protein A–coated gold particles are saturated with antibodies, there is no interaction between gold particles. Thus, multiple antigens can be determined even in single secretory granules. In fact, we demonstrated intragranular colocalization of immunoreactive oxytocin, labeled with 50-nm gold particles, and immunoreactive methionine-enkephalin, labeled with 15-nm gold particles, in the axonal terminals of the FD-prepared rat neurohypophysis. This study demonstrates the value of the use of gold-antibody complexes for immunocytochemical labeling on FS- or FD-treated tissues.