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Fluorescent Multiplex Immunohistochemistry

PD-L2 (orange), PD-L1 (E1L3N®) (red), CD68 (green), PD-1 (yellow), CD8α (magenta) and Pan-Keratin (cyan) on FFPE tonsil.

Our extensive validation and advanced technical support ensures that all of our IHC-P-validated antibodies can be used for fluorescent multiplex IHC.

Benefits of multiplex IHC

  1. Gather maximal data per tissue section, which is critical when tissue samples are limited.
  2. Understand co-expression and spatial organization of multiple targets within preserved tissue architecture, unlike alternative multiplex approaches (e.g., next generation sequencing, PCR, mass spectrometry, etc.

Being able to visualize multiple targets simultaneously is important in research fields like tumor immunology where it is necessary to catalog subsets of immune and cancer cells within the tumor microenvironment. For example, tumors may evade immune detection by manipulating the expression of immune checkpoint proteins (e.g., PD-1, CTLA-4, TIM-3) to “turn off” activated T cells. Alternatively, T cells may become less effective at destroying tumor cells as they succumb to exhaustion, a phenomenon characterized by changes in immune checkpoint protein expression, T cell expression of VISTA, and the appearance of CD68+ co-infiltrating macrophages. Thus, molecular profiling of immune checkpoint proteins together with immune cell phenotyping is key to understanding the complex tumor microenvironment and to enabling the development of tailored, combinatorial therapeutic intervention.

Multiplexing options

mIHC Multiplexing Options

Benefits of tyramide-based multiplexing:

  1. Signal amplification:
    • Provides marked enhancement of antigen-associated fluorescence signal.
    • Enables detection of low abundance targets.
  2. Simplified panel design: Compatible with any IHC-P validated antibody regardless of the host species or isotype.

Fluorescent mIHC with tyramide: overview

Tyramide is an organic phenol that, in the presence of a catalyst (HRP), becomes activated and covalently binds to electron rich regions, typically tyrosine residues, present on the surface or in the vicinity of protein antigens (see schematic below). Thus HRP-catalyzed deposition of tyramide-fluorophore molecules provides enhanced amplification of fluorescence signal at the site of the antigen.

Multiplex IHC Schematic

The covalent nature of tyramide-tyrosine engagement allows for heat-mediated removal (stripping) of primary/secondary antibody pairs bound to the antigen, while preserving the antigen-associated fluorescence signal. This facilitates the sequential use of multiple primary antibodies of the same host species or isotype without the concern for crosstalk, thereby greatly enabling multiplexing potential.

Requirements for success

Tyramide-based Fluorescent mIHC requires the following factors:

  1. Rigorously validated and highly specific primary antibodies (Abs) recommended for IHC-P.
    Note: all IHC-P validated Abs offered by CST are compatible with fluorescent mIHC.
  2. Secondary Abs specific to the host species/isotype of the primary Ab and conjugated to HRP, an enzyme that catalyzes the activation and permanent deposition of tyramide.
    Note: primary antibodies directly conjugated to HRP can also be used.
  3. Tyramide-fluorophore conjugates.
  4. Multispectral camera for mIHC panels that exceed 3 targets and a nuclear counterstain.

Multispectral versus Conventional imaging

The size of the antibody panel largely determines the imaging system of choice.

  • Small panel sizes (1-3 targets/fluorophores); conventional cameras are more than capable of providing robust resolution of each target in a low-plex context. See the comparison of wide-field images taken with a multispectral versus a conventional camera.
Multispectral vs. Conventional

PD-L1, CD3ε, CD8α Multiplex IHC Panel #65713: A 3-plex Fluorescent mIHC analysis of paraffin-embedded human breast cancer using PD-L1 (E1L3N®) XP® Rabbit mAb #13684 (green), CD3ε (D7A6E) XP® Rabbit mAb #85061 (yellow), and CD8α (C8/144B) Mouse mAb (IHC Specific) #70306 (Red). Blue pseudocolor = DAPI #8961 (fluorescent DNA dye).

  • Large panel sizes (more than 4 targets/fluorophores) mandate the use of multispectral cameras. Multispectral imaging systems support software enabling linear unmixing of fluorophores (and chromogens) that have overlapping excitation/emission spectra and thus promote multiplex target detection with superior resolution. This is critical particularly when co-expression analysis is to be performed.

Note: Tissue autofluorescence can be an issue often contributing to poor sensitivity in fluorescent IHC. This phenomenon is apparent in the context of all fluorescent imaging platforms. Multispectral analysis software addresses this issue by subtracting the artifactual fluorescence signal that originates from unstained tissue.