Multiplexed Tissue Staining with Signal Amplification

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.

• 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.

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.

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.

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).

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.

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.

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:

• ProLong Glass Antifade Mountant (Cat. No. P36984) is recommend for hard-set mounting and long-term archiving of tissue samples.
• SlowFade Glass Soft-set Antifade Mountant (Cat. No. S36917) is recommended for soft-set mounting if coverslip removal is required for tissue samples.
  For optimal results, follow the instructions provided with the mountant.

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.

Lin JR, Chen YA, Campton D, Cooper J, Coy S, Yapp C, Tefft JB, McCarty E, Ligon KL, Rodig SJ, Reese S, George T, Santagata S, Sorger PK. High-plex immunofluorescence imaging and traditional histology of the same tissue section for discovering image-based biomarkers. Nat Cancer. 2023 Jul;4(7):1036-1052. doi: 10.1038/s43018-023-00576-1. PMID: 37349501

PhenoImager is trademarked by Akoya Biosciences. BOND are trademarks of Leica Biosystems and its affiliates.