Fluorescent multiplex immunohistochemical analysis of paraffin-embedded human breast cancer using PD-L1 (E1L3N®) XP® Rabbit mAb (green), FoxP3 (D2W8E™) Rabbit mAb (IHC Specific) (yellow), and CD8α (C8/144B) Mouse mAb (IHC Specific) (Red). Blue pseudocolor = DAPI #8961 (fluorescent DNA dye). Image acquisition was performed with a multispectral camera.
Fluorescent multiplex immunohistochemical analysis of paraffin-embedded human tonsil using PD-L1 (E1L3N®) XP® Rabbit mAb (green), FoxP3 (D2W8E™) Rabbit mAb (IHC Specific) (yellow), and CD8α (C8/144B) Mouse mAb (IHC Specific) (Red). Blue pseudocolor = DAPI #8961 (fluorescent DNA dye). Image acquisition was performed with a multispectral camera.
|PD-L1 (E1L3N®) XP® Rabbit mAb||20 µl|
|CD8α (C8/144B) Mouse mAb (IHC Specific)||20 µl|
|FoxP3 (D2W8E™) Rabbit mAb (IHC Specific)||20 µl|
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).
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
Protocol Id: 1065
The PD-L1, FoxP3, CD8α Multiplex IHC Antibody Panel enables researchers to simultaneously detect these targets in paraffin-embedded tissues using tyramide signal amplification. Each antibody in the panel has been validated for this approach. For recommended staining conditions optimized specifically for this antibody panel please refer to Table 1 on the Data Sheet.
Each antibody in this panel recognizes endogenous levels of its specific target protein.
Monoclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human PD-L1 protein, recombinant protein specific to human FoxP3, or a synthetic peptide corresponding to residues near the carboxy terminus of human CD8α protein.
The field of cancer immunotherapy is focused on empowering the immune system to fight cancer. This approach has seen recent success in the clinic with targeting immune checkpoint control proteins, such as PD-1 (1,2). Despite this success, clinical biomarkers that predict response to therapeutic strategies involving PD-1 receptor blockade are still under investigation (3-5). While PD-L1 expression has been linked with an increased likelihood of response to anti-PD-1 therapy, research studies have shown that additional factors, such as tumor-immune infiltration and the ratio of effector to regulatory T cells within the tumor, could play a significant role in predicting treatment outcome (6-9).
Programmed cell death 1 ligand 1 (PD-L1) is a member of the B7 family of cell surface ligands that regulate T cell activation and immune responses. The PD-L1 ligand binds the PD-1 transmembrane receptor and inhibits T cell activation. PD-L1 is expressed in several tumor types, including melanoma, ovary, colon, lung, breast, and renal cell carcinomas (10-12).
FoxP3 is a transcription factor that is crucial for the development of T cells with regulatory properties (Treg) (13). Mutations in FoxP3 are associated with immune dysregulation, polyendocrinopathy, enteropathy, and X-linked syndrome (IPEX) (15), while overexpression in mice causes severe immunodeficiency (15). Research studies have shown that FoxP3 functions as a tumor suppressor in several types of cancer (16-18).
CD8 (Cluster of Differentiation 8) is a disulphide-linked heterodimer consisting of α and β subunits. On T cells, CD8 is the coreceptor for the TCR, and these two distinct structures recognize the Antigen–Major Histocompatibility Complex (MHC). CD8 ensures specificity of the TCR–antigen interaction, prolongs the contact between the T cell and the antigen presenting cell, and the α chain recruits the tyrosine kinase Lck, which is essential for T cell activation (19).
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