Introduction

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.


Materials

Procedural guidelines and tips
  • Do not let the tissue sample dry out. Dried out tissue will result in incorrect or no signal. Ensure that there is enough solution to completely cover the tissue during incubation and wash steps. We recommend using a humidified chamber (for example, a covered box with damp paper towel).
  • Crisp staining results require optimizing primary antibody dilution.
  • Optimization of the labeling step with Alexa Fluor tyramides or Aluora reagents is crucial for getting high resolution images which enhances signal to background.
  • If multiplexing fluorophores, please optimize the heat-induced epitope (antigen) retrieval (HIER) procedure for your samples.
  • Prepare reagents on day of use.

Procedure

Prepare reagents

Reagents included in the Tyramide SuperBoost kitReagents 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 streptavidinPoly-HRP-conjugated secondary antibody or HRP-conjugated streptavidin
Alexa Fluor tyramide reagentAluora dye
Hydrogen peroxideHydrogen peroxide
Reaction bufferReaction buffer
Reaction Stop ReagentReaction 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.

ReagentVolume
Alexa Fluor tyramide or Aluora dye

Entire content
DMSO
  • 150 μL for 150 slides kit size
  • 100 μL for 100 slides kit size
  • 50 μL for 50 slides kit size
  1. Dissolve the Alexa Fluor tyramide or Aluora dye in DMSO.
  2. Vortex to dissolve any reagent that might coat the sides or cap of the vial.
  3. Pulse briefly in centrifuge for contents to come down.

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.

ReagentVolume
Hydrogen Peroxide Solution1 drop
Distilled H2O1 mL
  1. Add 1 drop (approximately 50 µL) of Hydrogen Peroxide Solution to 1 mL of distilled water.

Note: Prepare the 100X H2O2 solution fresh on the day of use.

ReagentVolume
Reaction buffer1 drop
ddH2O1 mL
  1. Add 1 drop (approximately 50 µL) of 20X Reaction buffer to 1 mL of distilled water.

Note: Prepare the 1X reaction buffer fresh on the day of use.

11X reaction stop reagent stock solution

ReagentVolume
Reaction Stop Reagent1 vial
95% ethanol1.45 mL
  1. Add 1.45 mL of 95% ethanol to one vial of Reaction Stop Reagent.
  2. Vortex the vial to dissolve any stop reagent coating the sides and cap of the vial.

Note: Unused portion of the reaction stop reagent stock solution can be stored at –20°C for 6 months.

Reaction stop reagent working solution

ReagentVolume
11X reaction stop reagent stock solution10 μL
PBS100 μL
  1. In a separate tube, prepare reaction stop reagent working solution by diluting 1:11 in PBS before use to prepare a working solution.

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.

ComponentNumber of coverslips (18 mm × 18 mm)
11050
100X Alexa Fluor tyramide or Aluora dye stock solution1 μL10 μL50 μL
100X H2O2 solution1 μL10 μL50 μL
1X reaction buffer98 μL980 μL4.9 mL

Note: Prepare the working solution fresh on the day of use.

Sodium hydroxide (NaOH) stock solution

ReagentVolume
NaOH solution (50% w/w)0.5 mL
Deionized water9.5 mL

Working autofluorescence quenching solution

ReagentVolume
1M NaOH Stock2.4 mL
H2O2 (30% w/v)15 mL
PBS93.1 mL

Note: Final concentration should be 24 mM NaOH and 4.5% H2O2 in PBS.


Prepare humid chamber

Build a humidified chamber to keep samples from drying out.

  1. Wet paper towels with distilled water.
  2. Layer slide container with wet paper towels.
  3. Layer with Parafilm to cover the wet paper towels.


Prepare controls, optimize primary antibody dilution, and set tyramide labeling reaction

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.

  • If non-specific signal is present in negative controls or if the signal is blurry in positive controls, decrease the incubation time.
  • If dim or no signal is present in positive controls, increase the incubation time.

Control slides

  • No primary negative control (primary antibody omitted).
  • No primary and no secondary negative control (primary and secondary antibody omitted).


Tissue preparation

1. Dewax/deparaffinize and rehydrate the FFPE tissue according to standard IHC-P (paraffin) protocols.

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.


Quench endogenous peroxidase and block tissue

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.


Label with primary and poly-HRP secondary antibody

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.

  1. Label the tissue with primary antibody with mouse or rabbit as the host. Dilute the antibody in Blocking buffer.
    Note: We recommend optimizing the primary antibody staining concentration.
  2. Incubate with the tissue for 30-60 minutes at room temperature or overnight at 2–8°C in a humid environment.
    Note: If using a kit with Streptavidin, use a biotin-conjugated primary antibody.
  3. Wash the tissue for 2 minutes with 1X PBS at room temperature on a rocker. Repeat this step three times.
  4. Add 2–3 drops (approximately 100–150 µL) of poly-HRP-conjugated secondary antibody for unlabeled primary antibodies or HRP-conjugated streptavidin for biotinylated primary antibodies to the tissue.
  5. Incubate for 10-60 minutes at room temperature or overnight at 2–8°C in a humid environment.
    Note: If you observe non-specific signal, you can shorten this incubation period.
  6. Wash the tissue for 2 minutes with 1X PBS at room temperature on a rocker in a humid environment. Repeat this step three times.


Label with fluorophore(s)

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

  1. Apply 100 μL of the Alexa Fluor tyramide or Aluora dye working solution to the tissue.
  2. Incubate according to the optimized time (2–10 minutes) at room temperature in a humid environment.
    The reaction can be stopped (1) using reaction stop reagent or (2) by stripping antibodies by performing HIER. We recommend using HIER when multiplexing with primary antibodies.

    Using stop reagent; recommended for single reaction

  3. Apply 100 µL of reaction stop reagent working solution.
  4. Rinse the tissue three times with 1X PBS at room temperature.
  5. Kits containing Biotin-XX only: If using a kit containing Biotin-XX, then use conjugated streptavidin. See “Streptavidin conjugates recommended for the detection of Biotin-XX” table (below).
    Note:If using Biotin-XX with a fluorescent streptavidin, do not perform HIER after labeling with streptavidin, as that will also remove the fluorescent streptavidin.

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.

  1. Dilute Citrate Buffer (pH 6.0), Concentrate (Cat. No. 005000) 1:20 in distilled water.
  2. After completing the “Label with Alexa Fluor tyramide or Aluora dye” steps above, place the tissue in the diluted citrate buffer (pH 6.0) and heat in a microwave oven on 100% power until boiling (1–2.5 minutes).
  3. Reduce the power to 20% and keep microwaving for an additional 15 minutes.
  4. Let the tissue sample cool to room temperature while keeping it in the citrate buffer.
  5. Wash the sample twice with 1X PBS.
  6. Repeat the steps starting from "Block samples for non-specific binding" above with the next primary antibody of the same or different host species. Repeat these steps as needed for additional targets.

Counterstain and detect

1. Counterstain the tissue as needed using standard protocols. Some reagents recommended for nuclear counterstaining are listed below.

Counterstain reagents

2. Mount the coverslips using a mountant with antifade properties:

3. Analyze the tissue using a compatible imaging instrument.


How to pick secondary antibodies

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.


References

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.

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