DEMONSTRATION OF DESMOSOMAL ANTIGENS BY ELECTRON-MICROSCOPY USING CRYOFIXED AND CRYOSUBSTITUTED SKIN WITH SILVER-ENHANCED GOLD PROBE

In a previous post-embedding immunogold electron microscopic (EM) stud ies, localization of various desmosomal antigens was possible at high but not at low magnification. We developed a method for simultaneous d emonstration of epidermal desmosomal antigens at both low- and high-po wer EM magnifications by a method based on cryofixation and acetone cr yosubstitution and the use of a 1-nm gold probe with silver enhancemen t. Ultra-thin sections of Lowicryl K11M were incubated with primary an tibodies against desmoplakin, desmocollin, or desmoglein, followed by 1-nm gold-conjugated secondary antibody. Silver enhancement for 12 min provided the ideal labeling size for low-power visualization, whereas silver enhancement for 4-6 min was ideal for high-power EM observatio n. Each desmosome immunolabeled with the gold probe was dearly demonst rated, even at very low-power magnification. The level of background l abeling could be determined easily and the area of interest for high-p ower observation selected accurately. The fine ultrastructural appeara nce of desmosomal molecules was precisely demonstrated on high-power o bservation. This system should be useful for the immunocytochemical st udy of a variety of desmosomal antigens as well as other molecules of interest.