Tyramide signal amplification method in multiple-label immunofluorescence confocal microscopy

Division of Neuropathology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
Methods (Impact Factor: 3.22). 09/1999; 18(4):459-64. DOI: 10.1006/meth.1999.0813
Source: PubMed

ABSTRACT The tyramide signal amplification (TSA) method has recently been introduced to improve the detection sensitivity of immunohistochemistry. We present three examples of applying this method to immunofluorescence confocal laser microscopy: (1) single labeling for CD54 in frozen mouse brain tissue; (2) double labeling with two unconjugated primary antibodies raised in the same host species (human immunodeficiency virus type 1 p24 and CD68) in paraffin-biopsied human lymphoid tissue; and (3) triple labeling for brain-derived neurotrophic factor, glial fibrillary acidic protein, and HLA-DR in paraffin-autopsied human brain tissue. The TSA method, when properly optimized to individual tissues and primary antibodies, is an important tool for immunofluorescence microscopy. Furthermore, the TSA method and enzyme pretreatment can be complementary to achieve a high detection sensitivity, particularly in formalin-fixed paraffin-embedded archival tissues. Using multiple-label immunofluorescence confocal microscopy to characterize the cellular localization of antigens, the TSA method can be critical for double labeling with unconjugated primary antibodies raised in the same host species.

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Available from: Virawudh Soontornniyomkij, Jul 28, 2015
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    • "For immunofluorescent detection, slides were processed using the tyramide-amplification procedure (Stanarius et al., 1997, Stanarius et al., 1999, Toda et al., 1999, Wang et al., 1999, Buki et al., 2000, Bobrow and Moen, 2001). Briefly, the slides were incubated in affinitypurified rabbit anti-alarin (Santic et al., 2007) as primary antibody (1:100 in KPBS with 0.4% Triton-X) for 24 hr at room temperature. "
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    • "For KP double-labeling with either aromatase or GnRH, sections were processed using the described tyramide-amplification procedure to minimize the probability of cross-reactivity between the two rabbit polyclonal primary antibodies (Berghorn, 1994; Buki, 2000; Wang, 1999). The sections were incubated in KP primary antibody (1:1000 in KPBS with 0.4% Triton-X) for 48 hrs at 4°C. "
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    • "In some of our experiments we used two primary antibodies from rabbit for double labeling using modification of a protocol described previously (Hunyady et al., 1996; Shindler and Roth, 1996; Wang et al., 1999) . In this experiment a high-density labeling of a target protein for one immunofluorescent channel, was achieved through tyramide signal amplification (TSA) using the catalytic activity of horseradish peroxidase (HRP) whereas, the second target was then detected by sequential use of conventional secondary immunofluorescence labeling described previously. "
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