S U M M A R YThe tyramide signal amplification (TSA) technique has been shown to detect scarce tissue antigens in light and electron microscopy. In this study we applied the TSA technique at the electron microscopic level to pre-embedding immunocytochemistry. This protocol was compared to the non-amplified protocol. With the TSA protocol, the labeling of GM130, a cis -Golgi matrix protein, was tested in a cell line and found to be highly sensitive and more enhanced than that with the simple protocol. Moreover, the gold particles were well localized to the cis -side of the Golgi apparatus in both the TSA and the simple protocol. Since the tyramide signal amplification (TSA) technique was successfully introduced in immunoassays (Bobrow et al. 1989), this novel approach has widely been used for in situ hybridization and immunocytochemistry at the light (Köhler et al. 2000) and electron (Mayer and Bendayan 1997) microscopic levels. This technique is based on the ability of HRP, to which the antigenantibody complex binds indirectly, to catalyze the deposition of labeled tyramide onto proteins surrounding the HRP. This deposition is believed to take place at the site of the enzyme reaction, thus leading to good resolution.Here we report the use of the TSA method at the electron microscopic level using a pre-embedding nanogold-silver staining for visualization. A human melanoma cell line, G361, was purchased and cultured in RPMI supplemented with 10% FBS, and then fixed in 3.0% glutaraldehyde in 0.1 M phosphate buffer. The fixed cells were immersed in 1% sodium borohydride in PBS to block free aldehydes. Cells were quenched in 3% H 2 O 2 in 60% methanol to inactivate endogenous peroxidase and then incubated in blocking buffer (10% normal horse serum, 1% bovine serum albumin, 0.1% gelatin in PBS). With the simple protocol, cells were incubated in anti-GM130 Ab (1.0 g/ml or 0.1 g/ml) (BD Transduction Laboratories; San Diego, CA), biotinylated secondary antibody (1:200), and then streptavidin-nanogold (1:100) (Nanoprobes; Stony Brook, NY) in incubation buffer (10% normal horse serum, 1% bovine serum albumin, 0.1% gelatin in PBS). With the TSA protocol, the signal was amplified by the primary antibody (0.1 g/ml, anti-GM130 Ab), the biotinylated secondary antibody (1:200), streptavidin-HRP (1:500), biotinyl-tyramide (1:50) (PerkinElmer Life Sciences; Boston, MA), and the streptavidin-nanogold (1:100). Streptavidin-HRP was diluted in incubation buffer [10% normal horse serum, 1% BSA, 0.1% gelatin in TBS (0.15 M NaCl, 50 mM Tris-HCl, pH 7.5)] and biotinyl-tyramide diluted in the 1 ϫ amplification diluent (PerkinElmer). Next, cells underwent HQ silver enhancement (Nanoprobes) K E Y W O R D Styramide signal amplification pre-embedding immunocytochemistry
Photocatalyst has long been studied and used as a promising agent for reduction of air pollution, as it carries such useful characteristics as self-cleaning and powerful oxidation, and it leaves non-toxic by-products. Photocatalysis is a surface reaction and is initiated by absorbing light in the UV wavelength range, therefore the system efficiency is strongly dependent upon surface area and UV intensity. Until recently application of photocatalyst has been limited to low air flow rate with low concentration of pollutants since it has been very difficult for a compact system to provide the large surface area as well as the strong UV intensity. In this study, a very compact and innovative system with a honeycomb monolith substrate coated with photocatalyst and non- thermal plasma as an UV light generation source is suggested to obtain larger specific surface area and stronger UV intensity to effectively enhance the efficiency of photocatalysis. Using this system, parametric study has been made to determine the functional dependency of UV intensity and electric power consumption on such factors as supplied voltages, substrate lengths, air flow conditions and photocatalyst loading ratios.
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