To achieve high-resolution signal amplification in cellular and tissue applications, Molecular Probes is committed to the extensive development of tyramide signal amplification (TSA) in combination with our proprietary dyes and other detection technology. TSA — sometimes called CARD, for Catalyzed Reporter Deposition — is an enzyme-mediated detection method that utilizes the catalytic activity of horseradish peroxidase (HRP) to generate high-density labeling of a target protein or nucleic acid sequence in situ. The TSA method has been reported to increase the detection sensitivity up to 100-fold, as compared with conventional avidin–biotinylated enzyme complex (ABC) procedures. Moreover, for multiparameter detection of targets in either live or fixed cells or tissues, TSA can be combined with several of our other important technologies, including ChromaTide nucleotides, ULYSIS and ARES Nucleic Acid Labeling Kits (Labeling Oligonucleotides and Nucleic Acids - Section 8.2), primary and secondary antibodies, avidin and lectin conjugates (Antibodies, Avidins, Lectins and Related Products - Chapter 7), Enzyme-Labeled Fluorescence (ELF, Enzyme-Labeled Fluorescence (ELF) Signal Amplification Technology - Section 6.3), cytoskeletal stains (Probes for Cytoskeletal Proteins - Chapter 11), organelle probes (Probes for Organelles - Chapter 12), cell tracers and proliferation markers (Fluorescent Tracers of Cell Morphology and Fluid Flow - Chapter 14, Assays for Cell Viability, Proliferation and Function - Chapter 15) and receptor probes (Probes for Endocytosis, Receptors and Ion Channels - Chapter 16). Our Zenon Horseradish Peroxidase Antibody Labeling Kits (Zenon Technology: Versatile Reagents for Immunolabeling - Section 7.3, Molecular Probes' Zenon Labeling Kits - Table 7.14, Figure 7.56) are of particular utility when used in combination with TSA technology; see below for a description of our Zenon Antibody Labeling Kits enhanced with TSA technology (Z25090, Z25091).

TSA labeling is a combination of three (or four) elementary processes (Figure 6.5) that typically comprise:

• Binding of a probe to the target via immunoaffinity (proteins) or hybridization (nucleic acids) followed by secondary detection of the probe with an HRP-labeled antibody or streptavidin conjugate. Peroxidase conjugates of other targeting proteins such as lectins and receptor ligands are likely to be suitable for labeling targets, as is endogenous peroxidase activity. Unconjugated HRP is also useful as a neuronal tracer; its use in combination with TSA is demonstrated in .
• Activation of multiple copies of a labeled tyramide derivative by HRP. Most often a fluorescent or biotinylated tyramide has been used; however, labeling with other hapten-conjugated tyramides or with polymeric reagents, including tyramide-conjugated gold particles, has also been reported.
• Covalent coupling of the resulting highly reactive, short-lived tyramide radicals to residues (principally the phenol moiety of protein tyrosine residues) in the vicinity of the HRP–target interaction site, resulting in minimal diffusion-related loss of signal localization (Figure 6.6). In a unique application, fluorescein-labeled tyramine has been used to detect protein oxidation by reactive oxygen species (ROS, Generating and Detecting Reactive Oxygen Species - Section 18.2) in fibroblasts exposed to oxidative stress and in the extracellular proteins of endothelial cells exposed to an oxidative burst from phorbol myristate acetate–activated neutrophils.
• In direct TSA, the fluorescent signal can be immediately detected, resulting in both excellent spatial resolution (Figure 6.7, ) and high signal intensity. When using a hapten-labeled tyramide such as biotin-XX tyramide, the easily reversible DSB-X biotin tyramide or DNP-X tyramide (see below), a subsequent detection step is required using a bioconjugate that recognizes the hapten. This second detection step can include a dye-labeled hapten recognizer such as a fluorescent streptavidin (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6) or a fluorescent anti-hapten antibody (Anti-Dye and Anti-Hapten Antibodies - Section 7.4). Alternatively, the hapten-labeled tyramide can be detected using an alkaline phosphate– or HRP-labeled hapten recognizer in conjunction with a fluorogenic or chromogenic substrate (), resulting in another enzyme-amplified detection step. Chemiluminescent detection of an HRP-deposited biotin tyramide has also been reported. The streptavidin conjugate of NANOGOLD 1.4 nm gold clusters (N24918, Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6) has been used to make biotin tyramide conjugates visible in light and electron microscopy. Presumably, the antibody and streptavidin conjugates of Alexa Fluor FluoroNanogold 1.4 nm gold clusters (Secondary Immunoreagents - Section 7.2, Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6) can be used with hapten-labeled tyramides for correlated fluorescence, light and electron microscopy studies.

Figure 7.56 Labeling scheme utilized in the Zenon Antibody Labeling Kits. An unlabeled IgG antibody is incubated with the Zenon labeling reagent, which contains a fluorophore-labeled, Fc-specific anti-IgG Fab fragment (panel A). This labeled Fab fragment binds to the Fc portion of the IgG antibody (panel B). Excess Fab fragment is then neutralized by the addition of a nonspecific IgG (panel C), preventing crosslabeling by the Fab fragment in experiments where primary antibodies of the same type are present. Note that the Fab fragment used for labeling need not be coupled to a fluorophore, but could instead be coupled to an enzyme (such as HRP) or to biotin.

The signal amplification conferred by the turnover of multiple tyramide substrates per peroxidase label translates into practical benefits, namely ultrasensitive detection of low-abundance targets in fluorescence in situ hybridization, immunohistochemistry, neuroanatomical tracing and other applications. For example, we have utilized TSA and Alexa Fluor 488 tyramide to detect expression of low-abundance epidermal growth factor (EGF) and estrogen receptors by flow cytometry with far greater sensitivity than can be obtained using a directly labeled EGF probe (Figure 6.10) or fluorophore- or hapten-labeled antibodies to the estrogen receptor (Figure 6.11). Application of TSA resulted in significantly increased detectability of estrogen receptors in urinary bladder carcinomas, as compared with conventional immunohistochemical analysis.

Figure 6.10 Detection of epidermal growth factor (EGF) receptors directly or with signal amplification. Cells expressing high (A431 cells, panel A) and low (NIH 3T3 cells, panel B) levels of EGF receptors were either directly labeled with the preformed Alexa Fluor 488 complex of biotinylated epidermal growth factor (E13345, blue) or indirectly labeled with biotinylated EGF (E3477) followed by either Alexa Fluor 488 streptavidin (S11223, green) or HRP-conjugated streptavidin and Alexa Fluor 488 tyramide (purple), components of our TSA Kit #22 (T20932).

Figure 6.11 Enhancement of estrogen receptor detection sensitivity by tyramide signal amplification (TSA). SKBR3 cells with characteristically low levels of estrogen receptor expression were fixed, permeabilized and treated with H₂O₂ to inhibit endogenous peroxidase activity. A mouse anti–human estrogen receptor monoclonal antibody (Chemicon) was labeled with the Alexa Fluor 488 dye or with biotin using our Zenon Alexa Fluor 488 Mouse IgG₁ Labeling Kit (Z25002) or Zenon Biotin-XX Mouse IgG₁ Labeling Kit (Z25052), respectively. Detection of estrogen receptors using the labeled antibodies was performed on a Becton Dickinson FACScan flow cytometer with excitation at 488 nm. The cellular fluorescence intensity histograms represent detection with Alexa Fluor 488 dye–labeled antibodies (blue), biotinylated antibodies coupled to Alexa Fluor 488 streptavidin (S11223, green) and biotinylated antibodies coupled to HRP–streptavidin and Alexa Fluor 488 tyramide (TSA Kit #22, T20932; orange). The red histogram represents unstained cells.

TSA Kits

We have developed numerous TSA Kits that combine the versatile tyramide signal amplification technology with our high-performance Alexa Fluor, Oregon Green 488 and Pacific Blue tyramides, as well as with biotin-XX tyramide, DSB-X biotin tyramide and 2,4-dinitrophenyl (DNP)-X tyramide (Tyramide Signal Amplification (TSA) Kits - Table 6.1). Each kit provides sufficient materials to stain 50–150 slide preparations and includes the following components:

• Tyramide labeled with an Alexa Fluor dye, Oregon Green 488 dye or Pacific Blue dye, or with biotin-XX, DSB-X biotin or DNP-X
• HRP-conjugated anti–mouse IgG antibody, anti–rabbit IgG antibody or streptavidin
• Amplification reaction buffer
• H₂O₂ reaction additive
• TSA blocking reagent
• A detailed protocol for tyramide labeling (Tyramide Signal Amplification (TSA) Kits)

Our fluorescent dye– and hapten-labeled tyramides are not currently available as stand-alone reagents.

Zenon Horseradish Peroxidase Antibody Labeling Kits

Our Zenon Horseradish Peroxidase Antibody Labeling Kits, available for mouse IgG (Z25054, Z25154, Z25254), rabbit IgG (Z25354) and human IgG (Z25454), make it possible to quantitatively label even submicrogram quantities of a primary antibody with HRP immediately before it is applied to the sample (Zenon Technology: Versatile Reagents for Immunolabeling - Section 7.3, Molecular Probes' Zenon Labeling Kits - Table 7.14). Antibodies labeled with HRP using these Zenon Kits can replace the HRP–goat anti–mouse IgG and HRP–goat anti–rabbit IgG antibody conjugates in any of the TSA Kits containing these secondary detection reagents.

Zenon Antibody Labeling Kits Enhanced with TSA Technology

For mouse IgG₁ primary antibodies, we have developed the Zenon Mouse IgG₁ Labeling Kits enhanced with TSA technology (Z25090, Z25091), which provide the necessary reagents from both the Zenon Horseradish Peroxidase Mouse IgG₁ Labeling Kit and the corresponding Alexa Fluor TSA Kit, for researchers who want both the ease of labeling mouse IgG₁ antibodies with Zenon labeling reagents and the signal amplification afforded by TSA technology. We offer these enhanced Zenon Kits containing either the green-fluorescent Alexa Fluor 488 tyramide or the red-orange–fluorescent Alexa Fluor 568 tyramide (Z25090, Z25091). Each kit provides sufficient reagents for 25 labelings, including:

• Zenon HRP mouse IgG₁ labeling reagent
• Zenon mouse IgG blocking reagent
• Alexa Fluor 488 tyramide (in Kit Z25090) or Alexa Fluor 568 tyramide (in Kit Z25091)
• Dimethylsulfoxide (DMSO)
• TSA blocking reagent
• TSA amplification buffer
• Hydrogen peroxide (H₂O₂)
• A detailed protocol for Zenon complex formation and tyramide labeling (Enhanced Zenon(R) Mouse IgG Labeling Kits)

The Zenon HRP mouse IgG₁ labeling reagent contains Fab fragments of goat IgG antibodies directed against the Fc portion of intact mouse IgG₁ antibodies. These Fab fragments have been purified to ensure their selectivity for the Fc portion of the mouse IgG₁ antibody and then labeled with HRP. This Zenon HRP mouse IgG₁ labeling reagent is simply mixed with any mouse IgG₁ primary antibody to form the Fab–mouse IgG₁ complexes, which can be used for immunolabeling similar to that of primary antibodies covalently labeled with HRP. TSA technology is then used to detect the target-bound Fab–mouse IgG₁ complex. Each HRP label on the Fab–mouse IgG₁ complexes can activate multiple copies of the Alexa Fluor tyramide to produce short-lived tyramide radicals that are highly reactive with residues near the interaction site, yielding an amplified fluorescent signal with minimal diffusion.

Immunohistochemical Detection Using TSA

TSA detection can be applied to a variety of immunohistochemical specimen preparations, including crytostat sections, formaldehyde-fixed paraffin-embedded sections, plastic-embedded sections and cultured cells. In immunohistochemical applications (, ), sensitivity enhancements derived from TSA allow primary antibody dilutions to be increased — up to a 1:1,000,000 antibody dilution was possible in one reported case, although a 5- to 50-fold increase over the normal dilution factor is more common — in order to reduce nonspecific background signals. Additionally, the strong signal amplification provided by the TSA method can overcome relatively high autofluorescence of cells and tissues. Furthermore, because TSA and diaminobenzidine (DAB) oxidation are both peroxidase-mediated reactions, TSA is readily adaptable for correlated fluorescence and electron microscopy studies. In a very unique application, the significantly lower detection threshold of TSA, as compared with fluorescent secondary antibodies, allowed detection of two targets by primary antibodies raised in the same host species, without substantial crosstalk between the signals. The first target was detected using TSA and a primary antibody concentration that was so low that it was essentially undetectable by fluorescent secondary antibodies. The second target was then detected by conventional secondary immunofluorescence labeling.

Fluorescence In Situ Hybridization Using TSA

The increased sensitivity afforded by TSA (, Figure 6.14, Figure 8.92) can be critically important for detecting relatively short oligonucleotide probes and low-abundance mRNAs by fluorescence in situ hybridization (FISH). Cosmid detection in formalin-fixed, paraffin-embedded sections is cumbersome, and the ability to use smaller cosmid probes of less than 1000 bases in conjunction with TSA detection technology is likely to be an important technique for FISH. TSA is also faster than traditional FISH detection schemes, allowing definitive results to be obtained within a single day. In addition, a two-stage amplification method for ultrasensitive mRNA detection has been reported that combines TSA detection of biotinylated riboprobes with alkaline phosphatase–mediated fluorescence generation using Molecular Probes' unique ELF 97 phosphatase substrate (Enzyme-Labeled Fluorescence (ELF) Signal Amplification Technology - Section 6.3). TSA, however, is not a panacea for FISH sensitivity problems. Because both specifically and nonspecifically bound probe signals are amplified, TSA will not compensate for suboptimal hybridization conditions. Optimal probe concentrations are typically 2- to 10-fold lower for TSA-detected FISH than for conventional immunocytochemical detection procedures, again saving on the cost of expensive hybridization probes. Typically, FISH probes are labeled by indirect methods that use streptavidin- or antibody-conjugated HRP. Techniques for direct labeling of oligonucleotide probes have been developed to eliminate background signals due to nonspecific binding of peroxidase conjugates.

As with some other detection systems, TSA technology allows several probes to be hybridized simultaneously to identify multiple targets. Signal development using multicolored fluorescent tyramides must then be carried out sequentially, with a peroxidase inactivation step between each TSA reaction to prevent crosstalk (). TSA amplification followed by peroxidase inactivation through mild acid treatment with 0.01 M HCl for 10 minutes at room temperature and then reapplication of TSA using a fluorescent tyramide of a different fluorescent color has been used for at least triple-labeled in situ hybridization.

Figure 6.14 Digital image analysis comparison of in situ–hybridized biotinylated α-satellite probes detected using TSA Kit #23 (T20933) with HRP–streptavidin and Alexa Fluor 546 tyramide (right) or Alexa Fluor 546 streptavidin (S11225, left). Both images were converted to pixel intensity values using MetaMorph software (Universal Imaging Corporation) and transferred to a Microsoft Excel spreadsheet for plotting. Alexa Fluor 546 dye and DAPI (counterstain) intensity values are shown in red and blue, respectively. Alexa Fluor 546 dye intensity values below 35% of maximum were omitted for clarity.

Figure 8.92 Fluorescence in situ hybridization detected by tyramide signal amplification. Chromosome spreads were prepared from the cultured fibroblast cell line MRC-5 and hybridized with a biotinylated α-satellite probe specific for chromosome 17. The probe was generated by nick translation in the presence of biotinylated dUTP. For detection by TSA, hybridized chromosome spreads were labeled using TSA Kit #22 (T20932) with HRP-conjugated streptavidin and Alexa Fluor 488 tyramide (upper panel) or using TSA Kit #23 (T20933) with HRP-conjugated streptavidin and Alexa Fluor 546 tyramide (lower panel). After counterstaining with DAPI (D1306, D3571, D21490), images were obtained using filters appropriate for DAPI, FITC or TRITC.

Detection of Biotin-XX Tyramide, DSB-X Biotin Tyramide and Hapten-Labeled Tyramides

When a tyramide labeled with a hapten such as biotin-XX or the readily reversible DSB-X biotin (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6) is used for TSA in an indirect labeling technique, a signal-generation reagent or scheme is necessary. A fluorescent tyramide such as our Oregon Green tyramide can also be utilized as a hapten for subsequent detection and amplification by an anti-fluorescein/Oregon Green dye antibody (Anti-Dye and Anti-Hapten Antibodies - Section 7.4). Various reagents and reagent combinations have been reported for detecting enzyme-deposited biotin tyramide or fluorescein tyramide that should be equally suitable for use with our biotin-XX tyramide, DSB-X biotin tyramide, DNP-X tyramide or Oregon Green 488 tyramide, including:

• The streptavidin conjugate of alkaline phosphatase (S921, Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6) in combination with NBT/BCIP (N6495, B6492, N6547; Detecting Enzymes That Metabolize Phosphates and Polyphosphates - Section 10.3) or ELF 97 phosphate (E6588, Enzyme-Labeled Fluorescence (ELF) Signal Amplification Technology - Section 6.3)
• Our antibodies to the DNP chromophore (Molecular Probes' anti-hapten antibodies and conjugates - Table 7.20), which are described in Anti-Dye and Anti-Hapten Antibodies - Section 7.4
• The streptavidin conjugate of HRP (S911, Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6) or the rabbit anti-fluorescein/Oregon Green antibody conjugate of HRP (A21253, A21254; Anti-Dye and Anti-Hapten Antibodies - Section 7.4) in combination with a traditional chromogenic peroxidase substrate such as diaminobenzidine (DAB) ()
• Fluorescent conjugates of avidin or streptavidin, several of which are listed in Molecular Probes' selection of avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates - Table 7.23 (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6). Our Alexa Fluor 488 and Alexa Fluor 594 conjugates of anti-fluorescein/Oregon Green antibody (A11090, A11091, A11096; Anti-Dye and Anti-Hapten Antibodies - Section 7.4) can potentially be used to further amplify the signal (Figure 6.5) or shift the emission of the green-fluorescent Oregon Green 488 tyramide to red fluorescence
• Diaminobenzidine (DAB) Histochemistry Kits (D22185, D22187; Secondary Immunoreagents - Section 7.2), for direct use with biotin-XX tyramide and DSB-X biotin tyramide or conversion of fluorescent signals to permanent staining
• NANOGOLD and Alexa Fluor FluoroNanogold conjugates of antibodies (Secondary Immunoreagents - Section 7.2) and streptavidin (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6), for target localization using a combination of light and electron microscopy
• The streptavidin conjugate of Captivate ferrofluid (C21476, Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6), which can be utilized to efficiently separate low-abundance cellular targets amplified by biotin-XX tyramide or DSB-X biotin tyramide and the TSA technology. Binding of DSB-X tyramide–labeled targets to the Captivate ferrofluid is reversible under extremely mild conditions, potentially permitting isolation of fully viable rare cells using the unique Captivate microscope-mounted magnetic yoke assembly (C24700, Accessories for Fluorescence Microscopy and Magnetic Separation - Section 23.3) and disposable sample chambers (C24701, Accessories for Fluorescence Microscopy and Magnetic Separation - Section 23.3), which have been specially designed for use with the Captivate ferrofluid conjugates (Figure 7.55)

We have compiled several early references for biotin tyramide under the product number for our TSA Kit #21 (T20931). Some selected applications that have been reported for biotin tyramide that should work at least as well with our biotin-XX tyramide include:

• Fluorescence in situ hybridization (FISH) in paraffin wax–embedded sections of colon tumor cells
• mRNA detection of low-abundance cytokine genes using direct HRP conjugates of hybridization probes
• Detection of low–copy number sequences of human papillomavirus
• Combination of mRNA in situ hybridization and immunohistochemical detection
• Use of relatively short probes for DNA and mRNA FISH
• In situ PCR amplification of HIV-1 DNA in brain tissue, detection of HIV-1 p24 antigen in paraffin sections and detection of HIV-1 viral DNA integration sites using FISH
• Detection of low-abundance targets with mRNA probes in cryosections
• Generation of a color bar code for genes with the Fiber-FISH technique
• In situ hybridization in semi-thin plastic sections
• Demonstration of macrophages in histochemical sections
• Immunofluorescent labeling of GABA_A receptor subunits in the human brain
• β-Galactosidase (LacZ) detection in paraffin-embedded tissue sections
• Localization of the glial cell line–derived neurotrophic factor (GDNF) and its functional receptor in the dorsal root ganglion of rat nerves
• Endosome labeling using HRP-conjugated transferrin and biotin tyramide
• Detection of the distribution of nitric oxide synthase in developing embryonic rat brain, blood vessels of rat brain and bovine aorta
• Amplified detection of low-abundance targets in cultured neurites
• Anterograde tracing using HRP-conjugated lectins
• Conversion of a TSA-based detection method to an electron microscopy detection method

Previously cited applications of various fluorescent tyramides that are not available from Molecular Probes include:

• Fluorescence in situ hybridization (FISH)
• Double- and triple-color immunofluorescence studies using unconjugated primary antisera raised in the same species
• Detection of chromosome-specific repeat sequences
• Escherichia coli detection and speciation in water with a biotinylated peptide nucleic acid
• Detection of cyanobacteria in a highly autofluorescent sample with a singly labeled oligonucleotide probe
• Flow cytometric characterization of labeled bacteria
• Localization of the HuC and HuR genes in chromosomes by FISH
• Simultaneous detection of two neuropeptide receptors
• Immunocytochemical detection of low-level incorporation of 5-bromo-2'-deoxyuridine (BrdU, B23151; Assays for Cell Enumeration, Cell Proliferation and Cell Cycle - Section 15.4) in dividing cells ()
• Localization of a scarce leptin receptor using TSA in combination with ELF 97 phosphate (Enzyme-Labeled Fluorescence (ELF) Signal Amplification Technology - Section 6.3)

Anti-fluorescein/Oregon Green antibody conjugates of HRP (Anti-Dye and Anti-Hapten Antibodies - Section 7.4, Anti-fluorophore antibodies and their conjugates - Table 7.19) have been used with fluorescein-labeled probes and TSA to detect:

• Embryonic gene expression at the cellular level by FISH
• An mRNA probe for a calcium transporter protein
• A somatostatin receptor protein
• Tissue antigens, with a 10- to 100-fold increase in sensitivity over conventional staining methods
• mRNA in paraffin sections of organotypic multicellular spheroids

Double and Sequential Amplification with TSA

To achieve greater signal amplification, sequential rounds of amplification can be achieved using TSA (Figure 6.5) or TSA in combination with our ELF technology (Enzyme-Labeled Fluorescence (ELF) Signal Amplification Technology - Section 6.3), or using the permanent histochemical stains DAB (for HRP) or NBT/BCIP (for alkaline phosphatase). For example, in the first round biotin-XX tyramide can be deposited on a target using one of our biotin-XX tyramide TSA Kits (Tyramide Signal Amplification (TSA) Kits - Table 6.1). In a subsequent step the peroxidase conjugate of streptavidin that is used in TSA Kit #21 (T20931) is used again, but this time in combination with an Alexa Fluor tyramide, Oregon Green 488 tyramide, Pacific Blue tyramide or another round of biotin-XX tyramide. Presumably, this amplification can be continued for at least a third round, although some loss of spatial resolution may result. Biotin tyramide that has first been deposited at the binding site of a biotin-labeled riboprobe using the streptavidin conjugate of HRP has been further amplified with the streptavidin conjugate of alkaline phosphatase (S921, Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6) in conjunction with ELF 97 phosphate for the ultrasensitive detection of a scarce leptin receptor mRNA. In another example demonstrating the versatility of the TSA technology, several labeling technologies were combined to detect the HIV-1 virus:

• Hybridization with a 15-base peptide nucleic acid probe labeled with a single fluorescein dye
• Complexation with an HRP conjugate of anti-fluorescein antibody
• Incubation with biotin tyramide
• Incubation with the streptavidin conjugate of HRP
• Re-incubation with biotin tyramide
• Detection with the Alexa Fluor 488 conjugate of streptavidin (S11223, Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6)

Alternatively, for detection by light microscopy, the sample was incubated with the streptavidin conjugate of HRP in conjunction with DAB instead of Alexa Fluor 488 streptavidin.

DAB Histochemistry Kits

The use of HRP for enzyme-amplified immunodetection — commonly referred to as immunoperoxidase labeling — is a well-established standard histochemical technique. The most widely used HRP substrate for these applications is diaminobenzidine (DAB), which generates a brown-colored polymeric oxidation product localized at HRP-labeled sites. The DAB reaction product can be visualized directly by bright-field light microscopy or, following osmication, by electron microscopy. We offer DAB Histochemistry Kits for detecting mouse IgG primary antibodies (D22185) and biotinylated antibodies and tracers (D22187). Each kit contains:

• Diaminobenzidine (DAB)
• HRP-labeled goat anti–mouse IgG antibody (in Kit D22185) or streptavidin (in Kit D22187) conjugate
• H₂O₂ reaction additive
• Blocking reagent
• Staining buffer
• A detailed staining protocol (Diaminobenzidine Histochemistry Kits)

Each kit provides sufficient materials to stain approximately 200 slides.

Additional Tips on Using TSA Technology

Use of the TSA technology is not without its precautions. Among these is the possibility of endogenous peroxidase activity in certain cells, especially eosinophils. This activity can be at least partially blocked by incubation with 0.3–3% hydrogen peroxide for about 60 minutes. Second, when using biotin-XX tyramide or DSB-X biotin tyramide, endogenous biotinylated proteins are a potential problem (). Third, because of the significant signal amplification capability of TSA, nonspecific binding of labeled hybridization probes, antibodies and other targeting probes can lead to unacceptably high background staining. This can be alleviated to some degree with appropriate blocking reagents, and furthermore, the high sensitivity of TSA permits antibodies and nucleic acid probes to be highly diluted, far below the amount required for target saturation, thus reducing nonspecific background. Antibody and nucleic acid probe dilution can also substantially reduce the cost of an assay and the amount of an expensive or rare material required for staining.

Mammalian cells and tissues contain biotin-dependent carboxylases, which are required for a variety of metabolic functions. These biotin-containing enzymes produce substantial background signals when biotin–streptavidin detection systems are used to identify cellular targets (). Because the TSA technology is so sensitive, we recommended preblocking endogenous biotin in cells with our Endogenous Biotin-Blocking Kit (E21390, Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6) when using TSA Kits containing biotin-XX tyramide and the streptavidin conjugate of HRP. The Endogenous Biotin-Blocking Kit provides streptavidin and biotin solutions in convenient dropper bottles and an easy-to-follow protocol (Endogenous Biotin-Blocking Kit). Sufficient material is provided for approximately one hundred 18 mm × 18 mm glass coverslips.

Improvement of TSA detection by post-incubation heating has been reported. Addition of viscosity-increasing dextran sulfate, poly(vinyl alcohol), poly(ethylene glycol) or poly(vinyl pyrrolidone) to the medium is reported to decrease diffusion of the phenoxy radical intermediate, resulting in superior localization of the signal. Hybridization probes that are directly labeled with HRP are reportedly useful for lowering nonspecific binding when working with labeled tyramides. Endogenous peroxidase can be sufficient to yield labeling at the site of this activity in cells, as in the case of eosinophils. The review by Speel, Hopman and Komminoth gives additional practical suggestions and references.

TSA technology has been used successfully for over a decade. Many papers have been published that report the use of biotin tyramide for indirect labeling of targets or various fluorescent tyramides for direct labeling of targets. Direct labeling methods have the considerable advantage of saving a second step in the detection scheme. Moreover, labeling targets with fluorescent tyramides instead of biotin tyramide has the further advantage of avoiding amplification of endogenous biotin in cells and tissues, such as we have observed in mitochondrial staining with streptavidin conjugates in the absence of a biotinylated probe ().

We have compiled an extensive bibliography (Bibliography for T24831) that lists several suggested applications; continuously updated copies are available upon request from our Technical Assistance Department or through this web site. However, the biotin tyramide used in most of the early references did not have the additional 14-atom spacer that we utilize in our biotin-XX tyramide to make the probe more accessible to avidin conjugates. Furthermore, none of the specific fluorescent dyes or haptens in the early references are available in kits provided by Molecular Probes, so the specific methods described in these references should be considered guides rather than definitive protocols, and results using our TSA reagents may differ from those reported. In our experience, the Alexa Fluor 488 tyramide (Tyramide Signal Amplification (TSA) Kits - Table 6.1) provides greater signal and significantly greater photostability than fluorescein tyramide, and the other Alexa Fluor tyramides, Oregon Green 488 tyramide and Pacific Blue tyramide also yield intense staining of targets.