Article

Tyramide Signal Amplification Method in Multiple-Label Immunofluorescence Confocal Microscopy

Division of Neuropathology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
Methods (Impact Factor: 3.65). 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
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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 addition, although the secondary antibodies used for double labeling were both produced in rabbit, amplification of the p70S6K signal results in a complex that is only recognized by a biotinylated secondary antibody. Although intuitively, one would expect there to be some cross-reactivity between the secondary antibodies, it has been shown that when a primary antibody is used at a concentration that can only be detected by a fluorescent amplification method (we saw no detection without the amplification method), another primary antibody, raised in the same host species, can be used and demonstrated with a different flurochrome in subsequent conventional immunostaining of the same section since cross-reactivity is not an issue.3,10,38 "
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Questions & Answers about this publication

  • Virawudh Soontornniyomkij added an answer in Antibodies:
    What is the protocol in obtaining a double IHC with the same primary antibody host species?
    I've seen a few protocols doing a sequential double stain on human FFPE using primary antibodies from the same host species (i.e., www.ihcworld.com). Does anyone have a favorite or a simple method that works particularly well?
    Virawudh Soontornniyomkij
    The method I used was published long ago, see attached.
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      [Show abstract] [Hide abstract]
      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.
      Full-text · Article · Sep 1999 · Methods