A Pre-embedding Technique of Nanogold-Silver Staining for Immunocytochemical Localization of Gp100 in Cultured Melanocytes.
- Author:
Seung Won LEE
1
Author Information
1. Department of Anatomy, Chonnam National University Medical School, Korea. seunglee@chonnam.ac.kr
- Publication Type:Original Article
- Keywords:
Nanogold-silver staining;
Gold toning;
Gp100;
Pre-embedding IEM
- MeSH:
Glutaral;
Melanocytes*;
Melanosomes;
Membranes;
Microscopy, Immunoelectron;
Sensitivity and Specificity;
Silver;
Silver Staining
- From:Korean Journal of Anatomy
2002;35(1):75-82
- CountryRepublic of Korea
- Language:English
-
Abstract:
Nanogold-silver staining is a pre-embedding immunocytochemical technique, making it possible to label antigens with particulate markers at the electron microscopic level. This technique is based on the advancement of gold technology and on the development of silver staining or enhancement. In the present study, the method of Nanogoldsilver staining that includes the use of high concentration of glutaraldehyde and gold toning as well, was used to localize gp100 or a membrane bound antigen in melanosome of the cultured melanocytes. With the HMB45 antibody against gp100, biotinylated secondary antibody and streptavidin-nanogold followed by silver enhancement and gold toning led to highly specific labeling of gold particle over the melanosome compartments. The specificity of labelings obtained with this protocol was confirmed by the control experiment. Omission of the primary antibody led to very low background of labeling. The specific signal that appeared as a collection of gold particles was localized mainly to the peripheral part of melanosome. This finding provides the more detailed nature of gp100 localization in melanosome, which has not yet been shown by the previous immunocytochemical study such as the post-embedding immunoelectron microscopy.
