BRIEF REPORT |
Introduction of Tyramide Signal Amplification (TSA) to Pre-embedding NanogoldSilver Staining at the Electron Microscopic Level
Department of Anatomy, Chonnam National University Medical School, Gwangju, Korea (S-wL,SEL,SHK,EKH, KIN,KYA,CSB,BYK,SSP); Chonnam National University Dental Materials Research Institute, Gwangju, Korea (Y-JP); Department of Anatomy and Histology, Kurume University School of Medicine, Kurume, Japan (K-iN); and Department of Dermatology, National Kyushu Medical Center, Fukuoka, Japan (SI)
Correspondence to: Dr. Seung-won Lee, Dept. of Anatomy, Chonnam National University Medical School, Gwangju 501-746, Korea. E-mail: seunglee{at}chonnam.ac.kr
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Summary |
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Key Words: tyramide signal amplification pre-embedding immunocytochemistry
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 nanogoldsilver 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% H2O2 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 µg/ml or 0.1 µg/ml) (BD Transduction Laboratories; San Diego, CA), biotinylated secondary antibody (1:200), and then streptavidinnanogold (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 µg/ml, anti-GM130 Ab), the biotinylated secondary antibody (1:200), streptavidinHRP (1:500), biotinyltyramide (1:50) (PerkinElmer Life Sciences; Boston, MA), and the streptavidinnanogold (1:100). StreptavidinHRP 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 biotinyltyramide diluted in the 1 x amplification diluent (PerkinElmer). Next, cells underwent HQ silver enhancement (Nanoprobes) and gold toning followed by Epon infiltration. The sections were examined with a JEOL electron microscope.
First, we performed the simple protocol with the recommended use (1.0 µg/ml) of anti-GM antibody. The simple protocol led to gold particle labeling specific over the cis-side of Golgi apparatus (Figure 1A) . Omission of the primary antibody in the labeling protocol led to very low background labeling (Figure 1B). Preservation of the cellular ultrastructure was fairly good and many organelles, including nucleus, mitochondria, and Golgi apparatus, could be recognized.
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We found that a high concentration (3%) of glutaraldehyde can be used for fixation and for obtaining good sensitivity in immunocytochemistry without yielding any nonspecific binding (Kosaka et al. 1986). This can be done by the additional step of quenching (or free aldehyde blocking) with 1% sodium borohydride.
The issue of resolution with the TSA technique also seems to be mainly related to the working mechanism of tyramide. The gold particles with the TSA protocol, bound to the biotintyramide, are well localized within the cis-Golgi apparatus to a degree comparable to those with the simple protocols (Figures 3A3C). This finding is in accordance with the result that resolution of the TSA technique in post-embedding immunocytochemistry remains very good (Mayer and Bendayan 1997).
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Acknowledgments |
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Footnotes |
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Literature Cited |
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Bobrow MN, Harris TD, Shaughnessy KJ, Litt GJ (1989) Catalyzed reporter deposition, a novel method of signal amplification. Application to immunoassays. J Immunol Methods 125:279285[CrossRef][Medline]
Köhler A, Lauritzen B, van Noorden CJF (2000) Signal amplification in immunohistochemistry at the light microscopic level using biotinylated tyramide and nanogoldsilver staining. J Histochem Cytochem 48:933941
Kosaka T, Nagatsu I, Wu JY, Hama K (1986) Use of high concentrations of glutaraldehyde for immunocytochemistry of transmitter-synthesizing enzymes in the central nervous system. Neuroscience 18:975990[CrossRef][Medline]
Mayer G, Bendayan M (1997) Biotinyltyramide: a novel approach for electron microscopic immunocytochemistry. J Histochem Cytochem 45:14491454