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Demonstration of desmosomal antigens by electron microscopy using cryofixed and cryosubstituted skin with silver-enhanced gold probe

H Shimizu, T Masunaga, A Ishiko, T Hashimoto, DR Garrod, H Shida and T Nishikawa

Department of Dermatology, Keio University School of Medicine, Tokyo, Japan.

In a previous post-embedding immunogold electron microscopic (EM) studies, localization of various desmosomal antigens was possible at high but not at low magnification. We developed a method for simultaneous demonstration of epidermal desmosomal antigens at both low- and high-power EM magnifications by a method based on cryofixation and acetone cryosubstitution and the use of a 1-nm gold probe with silver enhancement. Ultra-thin sections of Lowicryl K11M were incubated with primary antibodies 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 observation. Each desmosome immunolabeled with the gold probe was clearly demonstrated, even at very low-power magnification. The level of background labeling could be determined easily and the area of interest for high-power observation selected accurately. The fine ultrastructural appearance of desmosomal molecules was precisely demonstrated on high-power observation. This system should be useful for the immunocytochemical study of a variety of desmosomal antigens as well as other molecules of interest.

Volume 42, Issue 5, pp. 687-692, 05/01/1994
Copyright © 1994 by The Histochemical Society


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X. Cheng, K. Mihindukulasuriya, Z. Den, A. P. Kowalczyk, C. C. Calkins, A. Ishiko, A. Shimizu, and P. J. Koch
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