Tyramide Signal Amplification Method in Multiple-Label Immunofluorescence Confocal Microscopy

METHODS 18, 459 – 464 (1999) Article ID meth.1999.0813, available online at http://www.idealibrary.com on

Tyramide Signal Amplification Method in MultipleLabel Immunofluorescence Confocal Microscopy Guoji Wang, Cristian L. Achim, Ronald L. Hamilton, Clayton A. Wiley, and Virawudh Soontornniyomkij Department of Pathology (Neuropathology), University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213

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 paraffinembedded 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. © 1999 Academic Press

Detection sensitivity may be an important limitation of immunohistochemistry. Formalin fixation and paraffin embedding, while preserving excellent histology, can adversely affect immunohistochemical detection of antigens. A variety of antigen retrieval procedures have been employed to unmask antigenic epitopes within tissues, e.g., enzyme pretreatment (trypsin, pepsin, pronase, proteinase K), alkaline hydrolysis, formic acid treatment, and high-temperature heating (1, 2). Recently, the tyramide signal amplification (TSA) method, originally described by Bobrow et al. as “catalyzed reporter deposition,” has been used to improve detection sensitivity in a variety of techniques 1046-2023/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

including enzyme immunoassays, Western blotting, in situ hybridization, and immunohistochemistry (3–9). This method has also been successfully applied to immunohistochemistry in formalin-fixed paraffinembedded autopsied brain tissue for detecting human immunodeficiency virus type 1 (HIV-1) p24 (10) and brain-derived neurotrophic factor (BDNF) (11). The amplification is achieved by biotin- or fluorophoreconjugated tyramide which acts as substrate for horseradish peroxidase (HRP). The HRP reacts with hydrogen peroxide and the phenolic portion of tyramide to produce highly reactive tyramide radicals that then covalently bind to electron-rich moieties (e.g., tyrosine) within tissues (3). Owing to the extremely short halflife of tyramide radicals, only tyrosine residues in close vicinity of the HRP will bind tyramide (5). The biotinconjugated tyramide can then be visualized by fluorophore- or enzyme-conjugated streptavidin. Primary antibodies can be applied for short incubation times or at low concentrations, resulting in decreased background. In general, both the biotin-conjugated and fluorophore-conjugated tyramides can significantly improve immunohistochemical detection sensitivity (6 – 8, 12). Multiple immunofluorescence (IF) labeling can usually be achieved with conventional methods using different fluorophore-conjugated secondary antibodies, if the primary antibodies used are raised in different host species. In detecting two unconjugated primary antibodies raised in the same species, recognition of both primary antibodies by each of the two different fluorophore-conjugated secondary antibodies is a major concern. With application of the TSA method, at a very low concentration of the primary antibody, the antigen cannot be detected by a conventional fluorophoreconjugated secondary antibody, but is detectable after 459

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FIG. 1. (A, B) Schematic representations of the conventional immunofluorescence method (A) and of the tyramide signal amplification method (B), modified with permission from the specification sheet of TSA-Direct (NEN Life Science Products, Boston, MA). (C, D) Immunofluorescence labeling of CD54 in frozen sections of ts-1 virus-infected mouse brain tissue. At the same concentration of biotinylated anti-CD54 antibody, the CD54 signal on vascular endothelia is barely visible with the conventional immunofluorescence method (C), but is distinctly visible with the tyramide signal amplification method (D). Bar 5 20 mm.

TYRAMIDE SIGNAL AMPLIFICATION IN CONFOCAL MICROSCOPY

TSA. Therefore, another primary antibody raised in the same host species can be applied and visualized with a different fluorophore in subsequent conventional IF labeling on the same tissue section (13, 14). According to Hunyady et al., successful double immunolabeling can be presumed when the first antigen can be clearly detected by the TSA method at a concentration of primary antibody at least 10-fold lower than that used in the conventional IF method (14). In our laboratory, we have successfully applied the TSA method to immunohistochemistry of neural and lymphoid tissues. Using this method, we are able to demonstrate the cellular localization of several antigens by immunofluorescence confocal microscopy in both frozen and paraffin tissues. Schematic representations of the conventional IF method and the TSA method are shown in Figs. 1A and 1B, respectively. Further, we present three examples of using the TSA method: (1) single IF labeling in frozen mouse brain tissue (Figs. 1C and 1D); (2) double IF labeling with unconjugated primary antibodies raised in the same host species in paraffin-biopsied human lymphoid tissue (Fig. 2); and (3) triple IF labeling in paraffinautopsied human brain tissue (Fig. 3).

DESCRIPTION OF METHOD Common Procedures Tissue sections were mounted on Fisherbrand Superfrost Plus glass slides (Fisher Scientific, Pitts-

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burgh, PA) to ensure adequate tissue adherence during immunohistochemical procedures. Specifications and sources of the primary and secondary antibodies and critical reagents used are listed in Tables 1 and 2. The commercially available kit Tyramide Signal Amplification (TSA-Direct, NEN Life Science Products, Boston, MA) was used in the experiments described below. TNB buffer (0.1 M Tris–HCl, pH 7.5, 0.15 M NaCl, 0.5% Blocking Reagent) and TNT buffer (0.1 M Tris–HCl, pH 7.5, 0.15 M NaCl, 0.05% Tween 20) were prepared according to the manufacturer’s protocols (NEN Life Science Products). The negative reagent controls were generated by replacing primary antibodies with normal sera from the same host species with equivalent protein concentrations. Following immunostaining, the sections were rinsed in phosphate-buffered saline (PBS, 3 3 5 min) and then mounted with Gelvatol. This water-soluble mounting medium can withstand the heat generated during laser-scanning microscopy. The “in-house” recipe for Gelvatol was modified from Current Protocols in Molecular Biology (15). Briefly, polyvinyl alcohol (20 g) was added slowly to PBS (100 ml) while constantly stirring [room temperature (RT), 4 – 6 h]. The solution was covered and under continuous stirring (4°C, overnight). Sodium azide (0.03 g) and glycerol (50 ml) were added and mixed thoroughly. The solution was then centrifuged at 10,000 rpm (4°C, 20 min), aliquoted into 10-ml syringes, and stored at 4°C until use.

FIG. 2. Double immunofluorescence labeling of HIV-1 p24 (with the tyramide signal amplification method, red) and CD68 (with the conventional method, green). Note that both unconjugated primary antibodies were raised in mice. Multinucleated giant cells in tonsillar lymphoid tissue from an HIV-1-infected individual, labeled with a macrophage marker CD68, express HIV-1 p24. Bar 5 20 mm. FIG. 3. Triple immunofluorescence labeling of BDNF (with the tyramide signal amplification method, green), GFAP (with the conventional method, red), and HLA-DR (with the conventional method, blue). A senile plaque in brain tissue from an individual with Alzheimer’s disease contains an HLA-DR-labeled microglial cluster and BDNF-labeled dystrophic neurites (arrowheads) within its core. GFAP-labeled astrocytes are located at the periphery of the plaque and extend their processes (arrows) into the plaque core. Bar 5 20 mm.

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Single Immunofluorescence Labeling for CD54 in Frozen Mouse Brain Tissue Conventional Method (Fig. 1C) Hemispheres of ts-1 virus (a temperature-sensitive mutant of Moloney murine leukemia virus)-infected mouse brains were fixed in 2% paraformaldehyde (2– 4 h) and then cryoprotected by immersing in 30% sucrose (4°C, 2 days). The tissues were frozen on dry ice and 20-mm-thick tissue sections were taken. After being rinsed in PBS (6 3 5 min), the sections were incubated with biotinylated hamster anti-mouse CD54 antibody (1:1000 dilution in PBS with 0.01% Tween 20, (4°C, overnight). After being rinsed in PBS (3 3 5 min), the sections were incubated with FITC-conjugated streptavidin (1:200 in PBS with 0.01% Tween 20) (RT, 1 h). Tyramide Signal Amplification (Fig. 1D) The frozen sections were rinsed in PBS (6 3 5 min) and then incubated with biotinylated hamster antimouse CD54 antibody (1:1000 in PBS with 0.01% Tween 20, (4°C, overnight). After being rinsed in PBS (3 3 5 min), the sections were incubated with TNB buffer (RT, 30 min), excess TNB buffer was blotted, and then the sections were incubated with HRPconjugated streptavidin (1:500 in TNB Buffer), (RT, 30 min). The sections were rinsed in PBS (3 3 5 min) and then incubated with FITC-conjugated tyramide (1:100 in 13 Amplification Diluent), (RT, 10 min). Double-Immunofluorescence Labeling for HIV-1 p24 and CD68 in Paraffin-Biopsied Tonsillar Lymphoid Tissue Using Unconjugated Primary Antibodies Raised in the Same Species (Fig. 2) Formalin-fixed paraffin-embedded sections were dewaxed (3 3 5 min), rehydrated in serial ethanol, treated with 3% hydrogen peroxide in methanol (30 min), rinsed in water, treated with 0.1% Pronase (37°C, 10 min), rinsed in water, rinsed in PBS (5 min), and incubated with 10% normal goat serum (RT, 20 min), and excess serum was drained. The sections were incubated with mouse anti-HIV-1 p24 antibody (1:100, (4°C, overnight). At this concentration of anti-HIV-1 p24 antibody, the HIV-1 p24 antigen could not be detected by Cy2-conjugated goat anti-mouse IgG serum (1:200, (RT, 1 h) in the conventional IF method (in which the optimal dilution of anti-HIV-1 p24 antibody was 1:5), but was still detectable after TSA. The sections were rinsed in PBS with agitation (3 3 5 min) and then incubated with biotinylated goat antimouse IgG serum (1:500, (RT, 30 min). After being rinsed in TNT with agitation (3 3 5 min), the sections were incubated with TNB buffer (RT, 30 min), excess TNB buffer was blotted, and then the sections were

incubated with HRP-conjugated streptavidin (1:500 in TNB Buffer, (RT, 30 min). The sections were rinsed in TNT with agitation (3 3 5 min) and then incubated with TRITC-conjugated tyramide (1:100 in 13 Amplification Diluent, RT, 10 min). After being rinsed in TNT with agitation (3 3 5 min) and in PBS (5 min), the sections were incubated with mouse anti-human CD68 antibody (1:100, RT, 2 h). The sections were rinsed in PBS (3 3 5 min) and then incubated with Cy2conjugated goat anti-mouse IgG serum (1:200, RT, 1 h). Triple Immunofluorescence Labeling for BDNF, GFAP, and HLA-DR in Paraffin-Autopsied Alzheimer’s Disease Brain Tissue (Fig. 3) Formalin-fixed paraffin-embedded sections were treated with the same protocol as described above for the lymphoid tissue, except that instead of being incubated with Pronase, the sections were immersed in preheated Target Retrieval Solution (95–99°C, 1 h). The sections were incubated with a mixture of rabbit anti-human BDNF antibody (1:400) and mouse anti-human HLA-DR antibody (neat, 4°C, overnight). At this concentration of anti-BDNF antibody, the BDNF antigen could not be detected by Cy3-conjugated goat anti-rabbit IgG serum (1:100, RT, 1 h) in the conventional IF method (in which the optimal dilution of anti-BDNF antibody was 1:20), but was still detectable after TSA. The sections were rinsed in PBS with agitation (3 3 5 min) and then incubated with biotinylated goat antirabbit IgG serum (1:200, RT, 30 min). After being rinsed in TNT with agitation (3 3 5 min), the sections were incubated with TNB buffer (RT, 30 min), excess TNB buffer was drained, and then the sections were incubated with HRP-conjugated streptavidin (1:400 in TNB buffer, RT, 30 min). The sections were rinsed in TNT with agitation (3 3 5 min) and then incubated with FITC-

TABLE 1 Primary Antibodies a Antibody to

Clone (isotype)

Mouse CD54 (ICAM-1) HIV-1 p24

3E2 (biotinylated hamster IgG) Kal-1 (mouse IgG1)

Human CD68 Human BDNF

PG-M1 (mouse IgG3) N-20 (rabbit IgG)

Bovine GFAP Human HLA-DR

(rabbit IgG) LN-3 (mouse IgG2b)

Manufacturer Pharmingen, San Diego, CA Dako Corp., Carpinteria, CA Dako Corp. Santa Cruz Biotechnology, Santa Cruz, CA Dako Corp. ICn Biomedicals, Aurora, OH

a ICAM-1, intercellular adhesion molecule-1; HIV-1, human immunodeficiency virus type 1; BDNF, brain-derived neurotrophic factor; GFAP, glial fibrillary acidic protein.

TYRAMIDE SIGNAL AMPLIFICATION IN CONFOCAL MICROSCOPY

conjugated tyramide (1:100 in 13 Amplification Diluent, RT, 10 min). After being rinsed in TNT with agitation (3 3 5 min) and in PBS (5 min), the sections were incubated with rabbit anti-bovine GFAP antibody (1:100, RT, 2 h). The sections were rinsed in PBS (3 3 5 min) and then incubated with a mixture of Cy5-conjugated goat anti-mouse IgG serum (1:100) and Cy3-conjugated goat anti-rabbit IgG serum (1:100, RT, 1 h). Confocal Microscopy The IF sections were analyzed with a Molecular Dynamics confocal laserscanning microscope (Sunnyvale, CA). This instrument is equipped with a Nikon inverted microscope with Plan-Apo 203 0.75-N.A. (air), 403 1.00-N.A. (oil), and 603 1.40-N.A. (oil) objective lenses. The illumination is provided by the argon/ krypton laser with 488-, 568-, and 647-nm primary emission lines. Each image was scanned along the z axis for a total of 10 sectional planes with a 0.5-mm step (512 3 512 pixels per sectional plane, 0.34 3 0.34 mm per pixel). Images were collected on a Silicon Graphics Inc. computer (Operating System Release 5.3, Farmington, MI) and analyzed using the Image Space software (Version 3.2, Molecular Dynamics). All multiplelabel IF images are 10-section projections. For triple labeling with FITC, Cy3, and Cy5 as an example, the specimen was first scanned for FITC and Cy5 signals. Using a 488/647 dual-bandpass laser wavelength filter, FITC and Cy5 were excited by the 488-nm line and the 647-nm line, respectively. Fluorescent light emitted by FITC and Cy5 passed through a 488/647 primary dual dichroic beamsplitter, and then was separated by a 650-nm secondary dichroic beamsplitter. Emitted light from FITC and Cy5 passed through a 530DF30 (between 515 and 545 nm) band-

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pass filter and a 660-nm long-pass filter, respectively. Subsequently, the specimen was rescanned for Cy3 signal with the same starting plane and ending plane along the z axis. Using a 568-nm bandpass laser wavelength filter, Cy3 was excited by the 568-nm line. Fluorescent light emitted by Cy3 passed through a 488/ 568 primary dual dichroic beamsplitter, and then passed through a 600DF40 (between 580 and 620 nm) bandpass filter. No secondary dichroic beamsplitter was used at this step.

CONCLUDING REMARKS The use of fluorophore-conjugated TSA can significantly improve the detection sensitivity in IF microscopy. Nevertheless, the variable reactivity of different markers with the TSA method underlines the necessity for individual testing of every primary antibody candidate to determine the optimal protocol for each type of tissue and fixation. With this method, both specific signal and nonspecific background are amplified. Important staining variables that need to be optimized include concentrations of primary antibodies, biotinylated secondary antibodies, HRP-conjugated streptavidin, and fluorophoreconjugated tyramide, incubation times, and temperature. In general, nonspecific binding of primary antibodies is diminished by the use of concentrations lower than those necessary to achieve comparable signal intensity with conventional IF methods (6). Consistent with the report of Adam et al. (6), we found that biotinylated secondary antibodies, although affinity-purified, can contribute to significant background, probably due to their nonspecific bind-

TABLE 2 Secondary Antibodies and Reagents Secondary antibodies and reagents a

Manufacturer

Biotinylated goat anti-mouse IgG (H 1 L) Biotinylated goat anti-rabbit IgG (H 1 L) Cy2-conjugated goat anti-mouse IgG (H 1 L) (excitation: 490 nm, emission: 508 nm) Cy3-conjugated goat anti-rabbit IgG (H 1 L) (excitation: 550 nm, emission: 570 nm) Cy5-conjugated goat anti-mouse IgG (H 1 L) (excitation: 650 nm, emission: 670 nm) FITC-conjugated streptavidin (excitation: 494 nm, emission: 517 nm) FITC-conjugated tyramide TRITC-conjugated tyramide (excitation: 550 nm, emission: 570 nm) HRP-conjugated streptavidin Amplification Diluent Pronase Target Retrieval Solution

Caltag Labs, San Francisco, CA Caltag Labs Jackson ImmunoResearch Labs, West Grove, PA Jackson ImmunoResearch Labs Jackson ImmunoResearch Labs Jackson ImmunoResearch Labs NEN Life Science Products, Boston, MA NEN Life Science Products NEN Life Science Products NEN Life Science Products Dako Corp., Carpinteria, CA Dako Corp.

a All secondary antibodies were affinity-purified. Note approximate peak wavelengths of excitation and emission for fluorophore-conjugated secondary antibodies. Cy3, indocarbocyanine; Cy5, indodicarbocyanine; FITC, fluorescein isothiocyanate; TRITC, tetramethylrhodamine isothiocyanate; HRP, horseradish peroxidase.

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ing to the tissue. We overcome this issue by optimizing the concentration of secondary antibodies, as well as by incubating the tissue sections (prior to the incubation with primary antibodies) with normal sera from the same species as secondary antibodies. To achieve a high detection sensitivity, particularly in formalin-fixed paraffin-embedded archival tissues, some antigen retrieval procedures may be needed to complement the TSA method (9 –11). The TSA method is a promising tool when conventional IF methods fail to detect specific signals. Confocal microscopy is an essential method in characterizing the cellular localization of antigens using multiple IF labeling in the same tissue section. Since in many circumstances the availability of primary antibodies is limited, the TSA method can be critical for double IF labeling with unconjugated primary antibodies raised in the same host species.

ACKNOWLEDGMENTS This work was supported by the NIH Grants NS35731 and MH46790 to C.L.A. and C.A.W.

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