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Multiplexing is essential for spatial biology studies as it allows for the simultaneous detection of multiple target molecules within a single tissue sample. The Tyramide SuperBoost technology is ideal for tissue multiplexing because it detects multiple proteins of interest through multiple rounds of applying and removing primary and secondary antibodies without significantly decreasing the fluorescence intensity of signal. Once antibodies are removed, the tissue can be reprobed with a primary antibody from mouse and rabbit without risk of cross-reactivity, followed by detection with another round of Tyramide SuperBoost signal amplification. By enhancing signal intensity and specificity, Tyramide SuperBoost Signal Amplification enables detection and visualization of multiple targets including low abundance proteins within tissue samples, contributing to a deeper understanding of spatial relationships and cellular interactions in biological systems.
How to: Invitrogen SuperBoost Tyramide Signal Amplification protocol
This protocol provides guidelines for utilizing the Tyramide SuperBoost kits for multiplex detection and visualization of target molecules in tissue starting from an unlabeled FFPE tissue sample on a slide to produce a multicolored tissue sample with multiple targets labeled with fluorescent tyramides. The labeled tissue is then ready for spatial imaging on any type of fluorescence microscope including the Invitrogen EVOS M7000 Imaging System and CellInsight CX7, or spatial imaging systems such as the Akoya PhenoImager. For added convenience, these steps can be performed on an automated slide stainer such as the Leica Bond RX systems.
Reagents included in the Tyramide SuperBoost kit
Note: Tyramide SuperBoost kits come in two sizes including 150 slides or 50 slides with or without secondary antibody conjugated to HRP.
Reagent | Volume |
---|---|
Alexa Fluor tyramide reagent (Component C1) | Entire content |
DMSO (Component E) | 150 μL (for 150 slides kit size) or 50 μL (for 50 slides kit size) |
You can store the 100X tyramide stock solution at 2–8°C for up to 6 months in a sealed vial. Store the vial away from moisture, if possible. For longer storage, aliquot into 5–10 μL volumes and store at –20°C.
Reagent | Volume |
---|---|
Hydrogen Peroxide Solution (Component C2) | 1 drop |
Distilled H2O | 1 mL |
Note: Prepare the 100X H2O2 solution fresh on the day of use.
Reagent | Volume |
---|---|
Reaction buffer (Component C3) | 1 drop |
ddH2O | 1 mL |
Note: Prepare the 1X reaction buffer fresh on the day of use.
11X reaction stop reagent stock solution
Reagent | Volume |
---|---|
Reaction Stop Reagent (Component D) | 1 vial |
95% ethanol | 1.45 mL |
Note: Unused portion of the reaction stop reagent stock solution can be stored at –20°C for 6 months.
Reaction stop reagent working solution
Reagent | Volume |
---|---|
11X reaction stop reagent stock solution | 10 μL |
PBS | 100 μL |
Note: Prepare the reaction stop reagent working solution fresh on the day of use.
The volumes in this table are based on 100 µL of tyramide working solution needed per 18-mm × 18-mm coverslip. This volume can be adjusted based on the size of the coverslip and tissue sample.
Component | Number of coverslips (18-mm × 18-mm) | ||
---|---|---|---|
1 | 10 | 50 | |
100X tyramide stock solution | 1 μL | 10 μL | 50 μL |
100X H2O2 solution | 1 μL | 10 μL | 50 μL |
1X reaction buffer | 100 μL | 1 mL | 5 mL |
Note: Prepare the tyramide working solution fresh on the day of use.
Sodium hydroxide (NaOH) stock solution
Reagent | Volume |
---|---|
NaOH solution (50% w/w) | 0.5 mL |
Deionized water | 9.5 mL |
Working autofluorescence solution
Reagent | Volume |
---|---|
1M NaOH Stock | 2.4 mL |
H2O2 (30% w/v) | 4.5 mL |
PBS | 93.1 mL |
Note: Final concentration should be 24 mM NaOH and 4.5% H2O2 in PBS.
Build a humidified chamber to keep samples from drying out.
We strongly recommend you optimize the primary antibody before using the kit. Positive and negative control slides should be stained with a serial dilution including 1:100, 1:500, 1:1000, 1:5,000, 1:10,000.
In many multiplex experiments, the primary antibody concentration can be optimized while keeping the secondary antibody staining and tyramide reaction conditions the same for all antibodies.
The incubation period for the tyramide working is crucial in getting high resolution images with specific signal. We highly recommend that you optimize the incubation period using positive and negative control slides at various incubation time points when conducting this experiment for the first time.
To optimize the incubation time for this step, perform 0, 2, 5, 7 and 10-minute incubations using positive and negative control slides.
Control slides
2. Perform heat-induced epitope retrieval (HIER) using either Citrate Buffer (pH 6.0) or EDTA (pH 9) using a microwave or pressure cooker according to standard antigen retrieval protocols.
Note: An automated slide stainer can be used for the dewaxing/deparaffinization and HIER steps.
(Optional) Perform autofluorescence reduction with white light prior to labeling (method described in Nat Cancer. 2023 Jul;4(7):1036-1052).
3. Rinse samples with 1X PBS at room temperature.
4. Place slides in a clear container covered with the working autofluorescence solution (4.5% hydrogen peroxide and 24 mM NaOH in PBS).
5. Illuminate with white light for 60 min.
Note: We do not recommend using the UV light as it can destroy certain antigens.
(Optional) Reduce non-specific dye binding with Image-iT FX Signal Enhancer
6. Rinse samples with 1X PBS at room temperature.
7. Apply 4 drops or 200 μL of Image-iT FX Signal Enhancer to cover each coverslip or section.
8. Incubate for 30 minutes at room temperature in a humid environment.
9. Rinse samples with 1X PBS at room temperature.
Prepare all reagents before starting these steps. Components A-D and C1-C3 are found in the kit.
Quench the endogenous peroxidase activity of the sample.
1. Cover the sample with 3% Hydrogen Peroxide Solution (Component C2).
2. Incubate for 60 minutes at room temperature in a humid environment.
3. Rinse samples with 1X PBS at room temperature.
(Optional) If using HRP-conjugated streptavidin secondary, block endogenous biotin in the sample. We recommend using the Invitrogen Endogenous Biotin-Blocking Kit (Cat. No. E21390).
4. Apply one or two drops of the streptavidin reagent (Component A from the Endogenous Biotin-Blocking Kit) to the cells or tissue and incubate for 15–30 minutes at room temperature in a humid environment.
5. Rinse samples with 1X PBS at room temperature.
6. Add one or two drops of the biotin reagent (Component B from the Endogenous Biotin-Blocking Kit) and incubate for 15–30 minutes at room temperature in a humid environment.
7. Rinse samples with 1X PBS at room temperature.
Block samples for non-specific binding.
8. Add 2–3 drops (approximately 100–150 µL) of Blocking buffer (Component A from the Tyramide SuperBoost kit) to the sample.
9. Incubate for 60 minutes at room temperature in a humid environment.
Note: If multiplexing using an automated slide stainer, the anti-mouse and anti-rabbit poly-HRP-conjugated secondary antibodies can be combined 1:1 into a single staining solution. This simplifies the procedure so that the same secondary antibody staining solution can be used when staining with either mouse or rabbit primary antibodies.
Prepare all reagents before starting these steps.
Complete optimizing both the tyramide working solution and the reaction stop reagent incubation periods before proceeding.
Label with tyramide
Performing antibody removal/stripping (HIER); recommended when multiplexing with primary antibodies from the same or different species.
For multiplexing on FFPE tissue samples, Tyramide SuperBoost kits are compatible with the citrate buffer/microwave method described by Tóth and Mezey (J Histochem Cytochem, 2007) to remove primary and secondary antibodies from the sample and eliminate peroxidase activity. This method does not significantly affect the fluorescence of the covalently reacted tyramide and allows the tissue to be reprobed with a primary antibody from either the same or a different species followed by subsequent Tyramide SuperBoost signal amplification.
Counterstain and detect
1. Counterstain the tissue as needed using standard protocols. Some reagents recommended for nuclear counterstaining are listed below.
Counterstain reagents
Counterstain type | Product | Ex/Em (nm) |
---|---|---|
Ready-to-use nuclear counterstains | NucBlue Fixed Cell ReadyProbes Reagent (Cat. No.R37606) | 360/460 |
NucGreen Dead 488 ReadyProbes Reagent (Cat. No.R37109) | 504/523 | |
NucRed Dead 647 ReadyProbes Reagent (Cat. No.R37113) | 642/661 | |
Concentrated nuclear counterstains | DAPI (Cat. No.D1306) | 358/461 |
SYTOX Green (Cat. No.S7020) | 504/523 | |
SYTOX Deep Red (Cat. No.S11381) | 660/682 |
2. Mount the coverslips using a mountant with antifade properties:
3. Analyze the tissue using a compatible imaging instrument.
SuperBoost TSA recommends using secondary antibodies conjugated to poly-HRP and are provided in the full kits. Secondary antibodies conjugated to HRP may not provide the optimal signal.
Biotin-XX Tyramide requires the use of fluorescent streptavidins, as listed in the table.
Streptavidin conjugates recommended for the detection of Biotin-XX tyramide | Ex/Em (nm) |
---|---|
Alexa Fluor 350 Streptavidin (Cat. No.S11249) | 346/442 |
Alexa Fluor 405 Streptavidin (Cat. No.S32351) | 402/421 |
Alexa Fluor 488 Streptavidin (Cat. No.S11223) | 495/519 |
Alexa Fluor 514 Streptavidin (Cat. No.S32353) | 518/540 |
Alexa Fluor 555 Streptavidin (Cat. No.S21381) | 555/565 |
Alexa Fluor 594 Streptavidin (Cat. No.S11227) | 590/617 |
Alexa Fluor 647 Streptavidin (Cat. No.S21374) | 650/668 |
Alexa Fluor 680 Streptavidin (Cat. No.S21378) | 679/702 |
Alexa Fluor 700 Streptavidin (Cat. No.S21383) | 702/723 |
Alexa Fluor 750 Streptavidin (Cat. No.S21384) | 749/775 |
PhenoImager is trademarked by Akoya Biosciences. BOND are trademarks of Leica Biosystems and its affiliates.
For Research Use Only. Not for use in diagnostic procedures.