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) 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.
Shimizu
H
H
Department of Dermatology, Keio University School of Medicine, Tokyo, Japan.
Masunaga
T
T
Ishiko
A
A
Hashimoto
T
T
Garrod
D R
DR
Shida
H
H
Nishikawa
T
T
D000328:Adult; D000941:Antigens / Q000032:analysis*; D015925:Cryopreservation; D003896:Desmosomes / Q000276:immunology / Q000648:ultrastructure*; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D016253:Microscopy, Immunoelectron / Q000379:methods*; D012867:Skin / Q000276:immunology / Q000648:ultrastructure*; D016610:Tissue Embedding
D000941:Antigens