The fluorescent simultaneous azo dye technique for demonstration of tartrate-resistant acid phosphatase (TRAP) activity in osteoclast-like multinucleate cells

Abstract

The purpose of our research was to show that the fluorescent simultaneous azo dye technique for tartrate-resistant acid phosphatase (TRAP) activity is useful for demonstration of osteoclasts under a fluorescent microscope and a confocal laser scanning microscope. Osteoclast-like multi-nucleate cells (MNC) were obtained from cultured mouse calvariae. The fluorescent simultaneous azo dye technique for TRAP activity was applied for cultured MNC. For the observation of specimens, both a conventional fluorescent microscope and a confocal laser scanning microscope for three-dimensional localization of TRAP-positive sites were used. Double staining with the azo dye technique was also adapted for mitochondria and tubrin staining. We found that the fluorescent simultaneous azo dye technique for TRAP activity could be used as a method of demonstrating MNC with a fluorescent microscope. We were also able to obtain the serial localization of three-dimensional sectioning images of TRAP-positive sites in MNC with a confocal laser scanning microscope. Moreover, with double staining of MNC as a modified version of the azo dye technique, we were able to observe both cell localization of TRAP-positive sites and other staining with epifluorescent illumination using the blue excitation method. Therefore, by using the fluorescent azo dye technique for TRAP activity, we can simultaneously observe not only the presence of osteoclasts, but also their characteristic construction and function.