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.
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.
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.
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.
NOTE: This step allows for the paraffin wax to melt.
NOTE: All washes are to be done with gentle agitation at room temperature.
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.
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.
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).
Cool slides to room temperature on bench top for 30 min.
Proceed with Staining/Detection (Step 5) using a different tyramide-fluorophore conjugate.
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.
posted April 2016