Amplify signals for fluorescence imaging

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Many functionally important proteins such as transcription factors and cell-surface cytokine receptors have native expression levels far below the detection threshold of labeled primary and secondary antibodies. Not only do cell signaling proteins vary in abundance by at least 7 orders of magnitude (~101 to 108 copies per cell), but their distribution within the cell is neither spatially nor temporally static. The Invitrogen SuperBoost kits with Alexa Fluor tyramides provide a highly sensitive method for detecting low-abundance targets in immunocytochemistry (ICC), immunohistochemistry (IHC), and in situ hybridization (ISH) applications. Tyramide SuperBoost technology combines the brightness of Alexa Fluor dyes with poly-HRP–mediated tyramide signal amplification to discern signal from noise (Figure 1), yielding precision and sensitivity 10–200 times greater than that of standard ICC/IHC/ISH methods and 2–10 times greater than that of other tyramide amplification techniques, including traditional TSA™ methods.

Tyramide signal amplification 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 [1]. In the first step of this process, a probe binds to the target via immunoaffinity (proteins) or hybridization (nucleic acids) and is then detected with an HRPlabeled secondary antibody or streptavidin conjugate. Next, multiple copies of a labeled tyramide (e.g., a fluorescent Alexa Fluor tyramide in the SuperBoost kits) are activated by enzymatic reaction with HRP. Lastly, the highly reactive, short-lived tyramide radicals covalently couple with 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 2).

Figure 1 (above). Detection of phosphorylated epidermal growth factor receptor (EGFR) using the Alexa Fluor 488 Tyramide SuperBoost Kit. A549 cells were incubated with epidermal growth factor (EGF) for 2 min to induce phosphorylation of EGFR on cell-membrane surfaces. Cells were fixed, permeabilized, and blocked according to the kit protocol. Phosphorylated EGFR was then labeled with rabbit anti-pEGFR primary antibody (clone Y1068) and detected using the Invitrogen Alexa Fluor 488 Tyramide SuperBoost Kit with goat anti–rabbit IgG antibody (green). Total EGFR (phosphorylated and unphosphorylated) was labeled with anti-EGFR primary antibody and detected with Invitrogen Alexa Fluor 594 secondary antibody (red). Nuclei were labeled with Invitrogen NucBlue Fixed Cell ReadyProbes Reagent (blue). Cells were mounted with Invitrogen ProLong Diamond mountant and imaged on an inverted confocal microscope.

Figure 2. Tyramide signal amplification applied to the immunolabeling of an antigen. The antigen is detected by a primary antibody (blue) (A), followed by a poly–horseradish peroxidase (poly-HRP) conjugated secondary antibody (yellow) (B). Activation of the dye-labeled tyramide (green) by HRP results in localized deposition of the activated tyramide derivative (pink) (C).

SuperBoost amplification provides greater sensitivity for hard-to-detect targets

The tyramide signal amplification technique used in the SuperBoost kits utilizes the catalytic activity of horseradish peroxidase (HRP) for high-density labeling of a target protein or nucleic acid sequence in situ. The enhanced sensitivity of the SuperBoost kits over other tyramide signal amplification technologies is in part due to improvements in the reagents for each amplification step. Figure 1 shows an example of the sensitivity of Tyramide SuperBoost technology to detect phosphorylated epidermal growth factor receptor (EGFR) in the cell membranes of A594 cells, just 2 minutes after treating cells with epidermal growth factor (EGF) and prior to receptor internalization.

The Tyramide SuperBoost kits boost the specific signal by utilizing Alexa Fluor tyramides, which react with HRP to deposit brightly fluorescent and photostable Alexa Fluor dyes on surrounding protein tyrosine residues and other similar molecules. In a study of cell-surface microdomains that serve as attachment sites for Kaposi’s sarcoma–associated herpesvirus (KSHV), Garrigues and coworkers reported that tyramide signal amplification with several different Alexa Fluor tyramides was 5 times more sensitive than conventional immunofluorescence in their experiments [2].

Furthermore, unlike traditional TSA kits, Tyramide SuperBoost kits employ poly-HRP– conjugated secondary antibodies (streptavidin is conjugated with standard HRP). Poly-HRP antibodies contain several HRP enzymes conjugated with short polymers, enhancing the signal several-fold over standard HRP antibodies (Figure 3); the molar ratio of enzyme to antibody is approximately 4. Importantly, the poly-HRP antibody is structured in such a way that the conjugate can penetrate cells or tissue as efficiently as do standard HRP antibodies.

Tyramide SuperBoost kits also employ several strategies to reduce background fluorescence. First, these kits contain highly cross-adsorbed secondary antibodies to help ensure specificity for the target primary antibody with minimal cross-labeling of other antibody species. For example, the poly-HRP–conjugated goat anti–mouse IgG exhibits no detectable reactivity to mouse serum proteins or IgG from bovine, goat, human, rabbit, or rat. Likewise, the poly-HRP–conjugated goat anti–rabbit IgG does not bind to rabbit serum proteins or IgG from bovine, goat, human, mouse, or rat. Second, an optimized blocking buffer is included to help prevent nonspecific binding and to significantly reduce endogenous peroxidase activity. And third, the Tyramide SuperBoost kits contain a reagent for preparing an HRP stop solution. Like any enzyme system, it is possible to overdevelop the signal, which can increase background levels. The HRP stop solution can be used to obtain maximum signal without an increase in background levels, which is especially important when amplifying signals localized to fine structures. Images produced with optimized HRP reaction times are as sharp as images produced with standard ICC/IHC/ISH methods but are 10 to 200 times more sensitive.

Figure 3. Sensitivity of Tyramide SuperBoost kits and other immunodetection methods using different amounts of primary antibody. HeLa cells were fixed and permeabilized with the Invitrogen Image-iT Fixation/Permeabilization Kit. Prohibitin was labeled with various dilutions of rabbit anti-prohibitin primary antibody; the manufacturer recommendation was 1:150 dilution or 5 μg/mL. Anti-prohibitin antibody was then detected using the Invitrogen Alexa Fluor 488 Tyramide SuperBoost Kit with goat anti–rabbit IgG antibody, the Invitrogen TSA™ Kit #12 with HRP goat anti–rabbit IgG antibody and Alexa Fluor 488 tyramide, or Invitrogen Alexa Fluor 488 goat anti–rabbit IgG secondary antibody. Cells were imaged on the Invitrogen EVOS FL Auto Imaging System using the same exposure and gain. These images (bottom) and the quantitation of fluorescence (top) indicate that the Alexa Fluor 488 Tyramide SuperBoost Kit is more sensitive than both the TSA kit and directly labeled secondary antibody. At this exposure and gain setting, prohibitin is not detectable with standard ICC methods.

SuperBoost amplification requires less primary antibody

Compared to conventional immunofluorescence techniques, the Tyramide SuperBoost kits require much smaller amounts of primary antibody—a tenth to a hundredth as much—to achieve sensitive detection of target molecules. Even with much less primary antibody, the Tyramide SuperBoost kits provide detection sensitivities similar to or greater than those obtained with a fluorescently labeled secondary antibody in an ICC application (Figure 3). Using less antibody per experiment will save on the cost of primary antibodies, a major expense in ICC and IHC workflows. Also, more experiments can be accomplished using a single vial of primary antibody. Given that some primary antibodies show significant lot-to-lot variation, using a single lot throughout a project can produce more reliable results that are consistent from experiment to experiment.

SuperBoost amplification is multiplexable

The Tyramide SuperBoost kits are available with four spectrally distinct Alexa Fluor tyramides and three different HRP secondary reagents (Table 1). This range of choices allows high-resolution detection and visualization of multiple signals in a single cell or tissue sample. In addition to multiplex detection using primary antibodies from different species (Figures 4A and 4B), Tyramide SuperBoost amplification is also compatible with experiments that use GFP and RFP fusions as reporters of gene expression (Figure 4C).

Unlike many other amplification methods, Tyramide SuperBoost technology allows you to detect multiple targets in tissues using primary antibodies from the same host species. Tyramide signal amplification produces highly reactive tyramide radicals that covalently react with tyrosine residues in the vicinity of the HRP conjugate, which results in minimal diffusion-related loss of signal and additionally makes it possible to strip off the primary and secondary antibody without significantly decreasing the fluorescence intensity of the tyramide deposit. Using the microwave/citrate buffer method described by Tóth and Mezey [3] to strip off antibodies, we validated this technique with tissue sections sequentially labeled with the Tyramide SuperBoost kits and three different rabbit primary antibodies (Figure 5) or two different rabbit primary antibodies (Figure 6).

Figure 4. Multiplexing Tyramide SuperBoost kits with other fluorescent probes. Prior to immunodetection, HeLa cells were fixed and permeabilized using the Invitrogen Image-iT Fixation/Permeabilization Kit. After staining, nuclei were labeled with Invitrogen NucBlue Fixed Cell ReadyProbes Reagent (blue) and then cells were imaged using a Zeiss LSM 710 inverted confocal microscope at 63x magnification. (A) Multiplexing Tyramide SuperBoost kits with secondary antibodies. In fixed and permeabilized HeLa cells, tubulin was labeled with rabbit anti-tubulin primary antibody and detected with Invitrogen Alexa Fluor 488 goat anti–rabbit IgG secondary antibody (green), and ATP synthase was labeled with mouse anti–ATP synthase subunit IF1 (ATPIF1) antibody and detected with Invitrogen Alexa Fluor 594 Tyramide SuperBoost Kit with goat anti–mouse IgG antibody (red). (B) Multiplexing two Tyramide SuperBoost kits. In fixed and permeabilized HeLa cells, prohibitin was labeled with rabbit anti-prohibitin primary antibody and detected using the Invitrogen Alexa Fluor 647 Tyramide SuperBoost Kit with goat anti–rabbit IgG antibody (purple), and β-catenin was labeled with mouse anti–β-catenin primary antibody and detected with Alexa Fluor 488 Tyramide SuperBoost Kit with goat anti–mouse IgG antibody (green). (C) Multiplexing Tyramide SuperBoost kits with a GFP reporter protein. Prior to fixation, HeLa cells were treated overnight with Invitrogen CellLight Peroxisome-GFP (BacMam 2.0) to produce a peroxisome-targeted GFP fusion protein (green). Cells were then fixed and permeabilized using the Image-iT Fixation/Permeabilization Kit. Prohibitin was labeled with rabbit anti-prohibitin primary antibody and detected using the Invitrogen Alexa Fluor 594 Tyramide SuperBoost Kit with goat anti–rabbit IgG antibody (red).

Figure 5. Sequential labeling and detection of three different rabbit primary antibodies using the Tyramide SuperBoost kits. A formalin-fixed, paraffin-embedded (FFPE) rat intestinal section was labeled sequentially with rabbit primary antibodies against H2B, actin, and Ki-67. Primary antibody detection was performed using three different Invitrogen Alexa Fluor Tyramide SuperBoost kits. Briefly, tissue samples underwent heat-induced antigen retrieval in citrate buffer, pH 6 (10 min on high setting in a pressure cooker) and were then sequentially labeled with rabbit anti-H2B antibody (detected with the Alexa Fluor 647 Tyramide SuperBoost Kit (green)), rabbit anti–smooth muscle actin antibody (detected with the Alexa Fluor 488 Tyramide SuperBoost Kit (red)), and rabbit anti-Ki67 antibody (detected with the Alexa Fluor 594 Tyramide SuperBoost Kit (blue)). In between each antibody labeling, tissue samples were microwaved in citrate buffer, pH 6, on high power until boiling (~2 min), then microwaved for 15 min at 20% power, and finally allowed to cool to room temperature before subsequent labeling with the next rabbit antibody [3]. Image was acquired on a Zeiss LSM 710 inverted confocal microscope at 20x magnification.

Figure 6. Same-species immunolabeling with Tyramide SuperBoost kits and detection of proliferation using the Click-iT EdU assay. Proliferating cells were detected in an FFPE rat mammary tissue section (derived from an animal that was pulsed with EdU) using the Invitrogen Click-iT EdU Alexa Fluor 647 Imaging Kit (pink). The sample was also labeled with two different rabbit primary antibodies (anti–Ki-67 and anti–smooth muscle actin) and two Tyramide SuperBoost kits (Alexa Fluor 594 Tyramide SuperBoost Kit for Ki-67, shown in white, and Alexa Fluor 488 Tyramide SuperBoost Kit for smooth muscle actin, shown in green) using the microwave/citrate buffer method [3] described in Figure 5. Images were acquired on a Zeiss LSM 710 inverted confocal microscope at 20x magnification.

Table 1. Tyramide SuperBoost kits.

Labeled tyramide Ex/Em * Cat. No. of Tyramide SuperBoost kits †
Goat anti–mouse IgG Goat anti–rabbit IgG Streptavidin
Alexa Fluor 488 495/519 B40912
B40941 (50 slides)
B40922
B40943 (50 slides)
B40932
Alexa Fluor 555 555/565 B40913 B40923 B40933
Alexa Fluor 594 591/617 B40915
B40942 (50 slides)
B40925
B40944 (50 slides)
B40935
Alexa Fluor 647 650/668 B40916 B40926 B40936
Biotin-XX NA B40911 B40921 B40931
* Approximate fluorescence excitation and emission maxima, in nm. † Sufficient reagents are provided for 150 18 mm x 18 mm coverslips using 100 µL per slide in critical incubation steps, except where otherwise noted. Poly-HRP–conjugated antibody and HRP streptavidin, as well as Alexa Fluor tyramides, are also available as stand-alone reagents.

Find out more about Tyramide SuperBoost amplification

Tyramide SuperBoost kits are designed for superior signal amplification, with the definition and clarity needed for high-resolution fluorescence imaging of low-abundance targets. These kits are simple to use and easily adapted to standard ICC, IHC, or ISH experimental protocols using a wide variety of cell and tissue types. We have tested the performance of the Tyramide SuperBoost kits using formaldehyde-fixed cells in 2D and 3D culture, formalin-fixed, paraffin-embedded (FFPE) tissues, and cryosection tissues.