Fluorescent Multiplex Immunohistochemistry (mIHC) with Tyramide Signal Amplification Protocol

A. Solutions, Reagents, and Kits

1. Xylene.
2. Ethanol, anhydrous denatured, histological grade (100% and 95%).
3. Deionized water (dH₂O).
4. Antigen unmasking solutions:

   1. Citrate: 10 mM Sodium Citrate, pH 6.0

      To prepare 500 ml 1X Citrate Unmasking Solution: add 50 ml SignalStain^® Citrate Unmasking Solution (10X) #14746 to 450 ml dH₂O.

   2. EDTA: 1 mM EDTA, pH 8.0

      To prepare 500 ml 1X EDTA Unmasking Solution: add 50 ml SignalStain^® EDTA Unmasking Solution (10X) #14747 to 450 ml dH₂O.

5. 3% Hydrogen Peroxide
   1. To prepare 500 ml 3% H₂O₂: add 50 ml of 30% H₂O₂ to 450 ml of dH₂O.
6. SignalStain^® Antibody Diluent (#8112).
7. Wash Buffer: 1X Tris Buffered Saline with Tween 20 (TBST)
   1. To prepare 1L 1X TBST: add 100 ml 10X TBST #9997 to 900 ml dH₂O.
8. SignalStain^® Boost IHC Detection Reagent (HRP rabbit, #8114; HRP mouse, #8125).
9. TSA Plus Fluorescein kit (Akoya Biosciences, NEL741001KT).
10. TSA Plus Cyanine 5 kit (Akoya Biosciences, NEL745001KT).
11. TSA Plus Cyanine 3 kit (Akoya Biosciences, NEL744001KT).
12. Stripping solution: 10 mM Sodium Citrate, pH 6.0
    1. To prepare 500 ml 10 mM Citrate solution: add 50 ml SignalStain^® Citrate Unmasking Solution (10X) #14746 to 450 ml dH₂O.
13. ProLong Gold Antifade Reagent with DAPI #8961.

B. Protocol overview

Fluorescent mIHC involving tyramide signal amplification (TSA) is a methodology that enables simultaneous detection of multiple proteins of interest in a given tissue section in a stepwise fashion. It is based on detection via indirect immunofluorescence involving primary and secondary antibodies to facilitate signal amplification.

In the protocol described below, an HRP-conjugated secondary antibody binds to an unconjugated primary antibody specific to the target/antigen of interest. Detection is ultimately achieved with a fluorophore-conjugated tyramide molecule that serves as the substrate for HRP. Activated tyramide forms covalent bonds with tyrosine residues on or neighboring the protein of interest and is permanently deposited upon the site of the antigen. This allows for serial stripping of the primary/secondary antibody pairs, while preserving the antigen-associated fluorescence signal, making this process amenable to multiple rounds of staining in a sequential fashion. Importantly, one of the key advantages of this method is that multiple primary antibodies of the same species can be used without the concern for crosstalk. This greatly simplifies and enables the process of a multiplex panel design.

C. Important tips

There are a number of considerations that can impact the success of a fluorescent multiplex IHC experiment involving tyramide.

Concentration of primary antibody: An optimal dilution of each primary antibody within a multiplex panel must be determined empirically and often can differ dramatically from the dilution recommended by the manufacturer due to the amplification of fluorescence signal afforded by tyramide deposition. We highly recommend optimizing the individual components of the multiplex panel by performing titrations for each component using a fluorophore of moderate intensity.

Order optimization: It is critical to optimize the order in which the antibodies in a multiplex panel are applied to the tissue section to ensure that multiple rounds of heating do not compromise target-specific epitopes. We recommend testing each optimized primary antibody within each slot of the multiplex panel using a fluorophore of moderate intensity to ensure that the fluorescence signal is not affected by the relative position within the panel.

Antibody-fluorophore pairing: Generally, it is good practice to pair antibodies detecting the lowest expressing targets with the brightest fluorophores. We recommend testing a matrix comprised of optimized primary antibodies and each available fluorophore in order to achieve the best possible signal intensity and signal to noise ratio for each target of a panel.

D. Protocol

1. Slide Baking (Optional)
   1. Incubate slides positioned horizontally for 1.5 hr at 60°C.
   2. Re-position the slides from a horizontal to an upright position and incubate for an additional 30 min at 60°C.

   NOTE: This step allows for the paraffin wax to melt.

2. Deparaffinization/Hydration
   1. Incubate sections in three washes of Xylene for 5 min each.
   2. Incubate sections in two washes of 100% ethanol for 10 min each.
   3. Incubate sections in two washes of 95% ethanol for 10 min each.
   4. Wash sections two times in dH₂O for 5 min each.

   NOTE: All washes are to be done with gentle agitation at room temperature.

3. Antigen Unmasking

   NOTE: Consult product data sheet for a recommendation on the optimal unmasking solution to use for each primary antibody in a multiplex panel. If the multiplex panel includes one antibody that is recommended for use with EDTA retrieval, use EDTA as the unmasking solution.

   For Citrate:

   1. Using a microwave, bring slides to a boil in 10 mM sodium citrate buffer, pH 6.0.
   2. Maintain at a sub-boiling temperature for 10 min.
   3. Cool slides to room temperature on bench top for 30 min.

   For EDTA:

   1. Using a microwave, bring slides to a boil in 1 mM EDTA, pH 8.0.
   2. Maintain at a sub-boiling temperature for 15 min. No cooling is necessary.
4. Quenching
   1. Wash sections in dH₂O three times for 5 min each.
   2. Incubate sections in 3% hydrogen peroxide for 10 min.
   3. Wash sections in dH₂O two times for 5 min each.
   4. Wash sections in 1X TBST for 5 min.
5. Staining/Detection

   NOTE: A separate pre-blocking of tissue sections may be performed but is not necessary. Optimal dilutions of the primary antibody must be determined empirically.

   1. Dilute primary antibody in SignalStain^® Antibody Diluent #8112.
   2. Add 100-400 μl to each section and incubate in a humidified chamber at room temperature for 60 min.
   3. During incubation with the primary antibody, equilibrate SignalStain^® Boost Detection Reagent (HRP rabbit, #8114 or HRP mouse, #8125) to room temperature.
   4. Remove antibody solution and wash sections with 1X TBST two times for 3 min each.
   5. Cover tissue sections in several drops (100-400 μl) of SignalStain^® Boost IHC detection Reagent (HRP rabbit, #8114 or HRP mouse, #8125) specific to the species of the primary antibody.
   6. Incubate in a humidified chamber at room temperature for 30 min, protected from light.
   7. Wash slides with 1X TBST two times for 3 min per wash, with gentle agitation and protected from light.
6. Tyramide Signal Amplification (TSA)

   NOTE: When choosing the appropriate fluorophore-conjugated TSA Plus amplification reagent, it is important to consider target expression levels and fluorophore intensity. Optimal pairing of primary antibody and fluorophore should be established in advance (see Important tips above).

   1. Dilute fluorophore-conjugated TSA Plus amplification reagent as per manufacturer’s recommendation.
   2. Apply 100-400 μl per slide and incubate for 10 min at room temperature in a humidified chamber, protected from light.
   3. Wash slides with 1X TBST two times for 3 min per wash, with gentle agitation and protected from light.
7. Serial staining
   1. If performing multiplex staining whereby more than one target of interest is to be detected:
      1. Proceed to stripping (Step 8).
   2. If you have completed your multiplex panel staining OR if you are performing a singleplex assay whereby a single target of interest is detected:
      1. Proceed to mounting (Step 9).
8. Stripping
   1. Using a microwave, bring slides to a boil in 10 mM sodium citrate buffer, pH 6.0.
   2. Maintain at a sub-boiling temperature for 10 min.

   3. Cool slides to room temperature on bench top for 30 min.

      Proceed with Staining/Detection (Step 5) using a different tyramide-fluorophore conjugate.

9. Mounting

   Mount sections with coverslips using ProLong Gold Antifade Reagent with DAPI #8961.

   NOTE: If slides are being used for the purpose of constructing a spectral library, ProLong Gold Antifade Reagent #9071 should be used.