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Multiplexing plays a crucial role in spatial biology studies as it enables the simultaneous detection of multiple target molecules within a single tissue sample. Enzyme-mediated signal amplification technology is particularly well-suited for tissue multiplexing due to the covalent attachment of the fluorophore. This allows for the detection of multiple proteins of interest through multiple rounds of applying and removing primary and secondary antibodies, without significantly reducing the fluorescence intensity of the signal.
It's important to note that Invitrogen SuperBoost kits are specifically designed for low-plex imaging applications in cells and tissues. These kits provide enhanced signal amplification, enabling increased sensitivity and improved detection of target molecules in samples. The Invitrogen Aluora Spatial Amplification Kits also enable enhanced signal amplification and are specifically developed for high-plex imaging (up to 8-plex) in FFPE tissue samples. These reagents facilitate the simultaneous detection and visualization of multiple target molecules within a single sample, contributing to a deeper understanding of spatial relationships and cellular interactions in biological systems.
How to: Invitrogen SuperBoost Tyramide Signal Amplification protocol
This protocol can be followed whether using Invitrogen SuperBoost or Aluora Spatial Amplification kits for detection and visualization of target molecules in tissue samples. This protocol starts from an unlabeled FFPE tissue sample on a slide and results in a multicolored tissue sample with multiple targets labeled with fluorescent dyes. The labeled tissue is then ready for spatial imaging on various fluorescence microscopes, including the Invitrogen EVOS M7000 Imaging System and CellInsight CX7 instrument, or spatial imaging systems such as the Akoya PhenoImager and EVOS S1000 system. For added convenience and efficiency during multiplexing, these steps can be performed using an automated slide stainer like the Leica Bond RX systems.
Reagents included in the Tyramide SuperBoost kit | Reagents included in the Aluora Spatial Amplification kit |
---|---|
Blocking buffer (10% Goat Serum) | Blocking buffer (10% Goat Serum) |
Poly-HRP-conjugated secondary antibody or HRP-conjugated streptavidin | Poly-HRP-conjugated secondary antibody or HRP-conjugated streptavidin |
Alexa Fluor tyramide reagent | Aluora dye |
Hydrogen peroxide | Hydrogen peroxide |
Reaction buffer | Reaction buffer |
Reaction Stop Reagent | Reaction Stop Reagent |
Note: Tyramide SuperBoost kits come in two sizes including 150 slides or 50 slides with or without secondary antibody conjugated to HRP. Aluora Spatial Amplification kits come in 100 slide kits.
Reagent | Volume |
---|---|
Alexa Fluor tyramide or Aluora dye | Entire content |
DMSO |
|
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 light and moisture, if possible. For longer storage, aliquot into 5–10 μL volumes and store at –20°C.
Reagent | Volume |
---|---|
Hydrogen Peroxide Solution | 1 drop |
Distilled H2O | 1 mL |
Note: Prepare the 100X H2O2 solution fresh on the day of use.
Reagent | Volume |
---|---|
Reaction buffer | 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 | 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 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 Alexa Fluor tyramide or Aluora dye stock solution | 1 μL | 10 μL | 50 μL |
100X H2O2 solution | 1 μL | 10 μL | 50 μL |
1X reaction buffer | 98 μL | 980 μL | 4.9 mL |
Note: Prepare the 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 quenching solution
Reagent | Volume |
---|---|
1M NaOH Stock | 2.4 mL |
H2O2 (30% w/v) | 15 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-based reaction conditions the same for all antibodies.
The incubation period for the Alexa Fluor tyramide or Aluora dye 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 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.
Quench the endogenous peroxidase activity of the sample
1. Cover the sample with 3% Hydrogen Peroxide Solution.
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 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 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 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 Alexa Fluor tyramide or Aluora dye working solution and the reaction stop reagent incubation periods before proceeding.
Label with Alexa Fluor tyramide or Aluora dye
Performing antibody removal/stripping (using HIER); recommended when multiplexing with primary antibodies from the same or different species
For multiplexing on FFPE tissue samples, SuperBoost and Aluora Spatial Amplification 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 attached fluorophore and allows the tissue to be reprobed with an additional primary antibody from either the same or a different species followed by subsequent SuperBoost or Aluora 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.
Signal amplification protocols recommend 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.