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Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis
Published on: December 15, 2011
Demonstration of desmosomal antigens by electron microscopy using cryofixed and cryosubstituted skin with
H Shimizu1, T Masunaga, A Ishiko
1Department of Dermatology, Keio University School of Medicine, Tokyo, Japan.
Insights
A new method enables simultaneous visualization of desmosomal antigens at both low and high electron microscopic magnifications. This technique improves immunolabeling for studying desmosomal molecules and ultrastructure.
Area of Science:
- Cell Biology
- Immunoelectron Microscopy
- Molecular Biology
Background:
- Previous electron microscopic (EM) studies limited desmosomal antigen localization to high magnifications.
- Effective simultaneous low- and high-power EM visualization of desmosomal antigens was lacking.
Purpose of the Study:
- To develop a novel method for simultaneous demonstration of epidermal desmosomal antigens at both low- and high-power EM magnifications.
- To enable precise ultrastructural analysis of desmosomal molecules.
Main Methods:
- Cryofixation and acetone cryosubstitution technique.
- Utilized a 1-nm gold probe with silver enhancement for immunolabeling.
- Incubation of ultra-thin Lowicryl K11M sections with primary antibodies (desmoplakin, desmocollin, desmoglein) followed by secondary antibody.
Main Results:
- Simultaneous demonstration of desmosomal antigens at both low- and high-power EM magnifications was achieved.
- Optimized silver enhancement times (12 min for low-power, 4-6 min for high-power) provided ideal labeling.
- Desmosomes were clearly visualized even at very low magnifications, with accurate selection of areas for high-power observation.
Conclusions:
- The developed method allows clear visualization of desmosomal antigens across magnifications.
- This technique facilitates accurate ultrastructural analysis of desmosomal molecules.
- The system is broadly applicable for immunocytochemical studies of desmosomal antigens and other molecules.
Abstract:
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.
Related Concept Videos
01:05Desmosomes
01:20Preparation of Samples for Electron Microscopy
01:20Immunogold Electron Microscopy