Application of Tyramide Signal Amplification (TSA) Both to Biochip Platform and to the Immunoelectron Microscopy to Label Proteins within the Organelle

Hong, Eun Kyoung; Lee, Song Eun; Jung, Chaeyong; Ahn, Kyu Youn; Bae, Choon Sang; Kim, Baik Yoon; Park, Sung Sik; Lee, Seung Won

Abstract

The tyramide signal amplification (TSA) technique, based on the ability of HRP to catalyze the deposition of tyramide onto the surrounding proteins, has been proved to detect scarce tissue antigens. In this study we applied this technique to a biochip platform and an immunocytochemistry at the electron microscopic level. First, in the optical fluorescence sensing, the signal was amplified by Dako Envision(TM) (goat anti-mouse immunoglobulins IgG conjugated to peroxidase labelled-dextran polymer) and tyramide-Cy3, which was then compared to the non-amplified control using goat antimouse IgG-Cy3 conjugate instead. The result showed that the tyramide method produced a more sensitive signal than the control method. Secondly, in the pre-embedding immunocytochemistry, we investigated to see whether it is possible to label proteins within a organelle in the cell using the TSA method. The signal was amplified by a primary antibody, a biotinylated secondary antibody, streptavidin-HRP, biotinyl-tyramide, and streptavidinnanogold followed by silver enhancement and gold toning. Then, this protocol was compared to the non-amplified or simple protocol that does not include the steps of streptavidin-HRP and biotinyl-tyramide. With the TSA protocol, the labeling for a membrane bound antigen (gp100) that is known to be exclusively localized to melanosomes in melanocyte, was tested in a melanoma cell line (G361) and found to be highly sensitive and more enhanced than with the simple protocol. Moreover, the gold particles were well localized to the subcellular structures or melanosomes both in the TSA and simple protocols, which indicates that resolution of the signals remains high. Control experiment with omission of the primary antibody demonstrated that background levels or nonspecific bindings are negligible. This result showed that the TSA method can be successfully applied to label the intra-organelle protein that is known to be labeled only in the specific fixation condition with the optimal permeability.