Tyramide can be essentially considered as a HRP substrate in a normal IHC/ ISH protocol. As the enzyme acts on the tyramide it deposits in the location of the antigen detected. Whereas systems such as ABC or fluorophore conjugates have limitations as to the size they can be before they fall out of solution, the deposition of tyramide in situ does not need to follow these constraints. Thus, more reporting molecules can be deposited at the site, and the signal boosted by several orders of magnitude.
Whilst more sensitivity is often a good thing, this also means that any background on a sample is also amplified by several orders of magnitude too! It is worth running controls during the optimisation to ensure that sources of background, such as endogenous peroxidase or off-target antibody binding are eliminated or reduced as much as possible. For normal IHC, there’s usually negligible background at around 6% antibody cross reactivity, but a sensitive tyramide stain may have issues even with less than 1% cross reactivity.
Reagents Required
Blocking
Serum block
Peroxidase blocking solution – for endogenous peroxidase
Biotin blocking kit (for biotin based methods)
HRP Antibodies e.g. Directly labelled, Via ABC reaction or ImmPRESS Polymer reagents
Tyramide reagent– Fluorophore or Biotin
Stop solution PBS/BSA/NaN3 – the azide kills the HRP reaction. Other HRP quenching methods may be possible. A heat treatment, such as an antigen unmasking step can also stop the reaction, and is also used as an antibody stripping step when multiplexing (see later)
Suggested Reaction buffer: - TRIS buffer, pH7.4
Outline IHC Method (excluding wash steps) :-
- Deparaffinize/ de-wax sections & re-hydrate (FFPE only)
- Antigen unmasking – as required
- Block endogenous HRP
- Serum block
- Block endogenous biotin (if using biotin system)
- Incubate with primary ab
- Primary antibody detection – HRP/ HRP Polymer secondary or biotin secondary & ABC detection as appropriate
- Incubate with Tyramide & use Stop solution
- If using Biotin Tyramide – apply final detection reagent(s)
- Mount appropriately. ( i.e. dehydrate & mount for non-aqueous HRP substrates, or aqueously for aqueous substrates & fluorophores) For fluorescent detection, an antifade mounting media is recommended.
Optimal dilutions and timings to be determined empirically.
Optimising:-
As for other IHC experiments, working sequentially back through the method from the end detection reagent enables identification of which layer in the method is causing non-specific staining. By ensuring each control is clean before moving on to the next step, any introduced background is likely caused by the new step added into the test.
- If using a colorimetric substrate try this on the tissue alone first after enzyme blocking. Increase enzyme blocking until clean
- Try biotin reagent + detection on sections alone to check for endogenous biotin.
- For fluorescent signals, check for autofluorescence.
- Try tyramide & subsequent detection on sections alone – re-check HRP blocking if required
- Secondary antibody, tyramide & detection
- Primary antibody tyramide & detection – An isotype control can be useful here to demonstrate staining is a result of the primary antibody is binding to the tissue, not non-specific Ig reactions, but does not prove primary antibody specificity for the target.
Although autofluorescence may be blocked by some kits at the end of staining, just before mounting, this protocol may also need to be optimised with tyramide. The quenching reaction can cause issues with the tyramide stain, in addition to quenching fluorophores slightly, so the quenching reagents may need to be diluted to enable the quenching reaction to be consistently controlled through exposure time.
Multiplexing.
The tyramide is activated by the HRP and enables it to bind to tyrosine on the tissues. Unlike the antibodies, this withstands a heat treatment such as unmasking. In this way, several rounds of directly fluorescent tyramide signals can be built on a section and cross reactivity issues between primary and secondary antibodies are avoided as the antibodies are removed between each stain by a heat treatment. However, care must be taken to optimise each layer individually, including the number of heat treatments antigens can withstand before they deteriorate too. Staining sequences for best results need determining empirically.