
Bringing the Tumor Microenvironment into Focus:
Simplified Development of Seven-Color Multiplex Immunohistochemistry-Immunofluorescence (mIF) Panels
With the advancement of immunotherapeutics, the need to understand the tumor microenvironment has never been more pressing. Recent advances in mIF and multispectral imaging facilitate accurate simultaneous analysis of multiple tissue markers.
Melissa Whiteman, PhD, Eric McIntush, PhD, Michael Spencer, PhD
Development time of a seven-color mIF was reduced using IHC validated antibodies and the optimized DAB dilution. Antibody order was guided by results of three slides stained after first, third or sixth HEIR. The ratio of target staining/DAPI nuclear counts, average intensity and overall background reveals the optimal order of staining. Some targets reveal optimal staining with greater intensity and lower background when stained last, such as FOXP3 (Step 2, Table 1 and Figure 1). For other antibodies, the opposite may be true or there could be no effect from multiple HIER. There are 720 possible combinations for a seven-color panel. Using this method, the number of slides was reduced to three per target (18) plus confirmation seven-color slides resulting in a panel containing CD3, CD8, CD68, Cytokeratin, FOXP3 and PD-L1 (Step 5, Figure 2).
Multiplex IF is a powerful technique that allows for examination of spatial arrangement of proteins of interest as well as protein interaction/co-localization of multiple targets within a single tissue specimen. mIF panels can take eight or more weeks to optimize, however, researchers can save time and resources using validated antibodies and this antibody order guide.
Provides colocalization and spatial orientation of proteins, which facilitates an accurate determination of the target’s subcellular localization, identification of multiple cell types, and resolution of the relative proximity of biomarkers
Obtain information on the expression levels of biomarkers while increasing the number of
biomarkers that can be visualized simultaneously on a single slide
Capture superior information about the tissue microenvironment
mIF is performed on formalin-fixed paraffin embedded tissue sections

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TSA multiplexing allows the user to choose any rabbit or mouse antibody without cross-reactivity: Select the best antibodies!
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Step 1
Select high quality IHC validated antibodies and confirm dilution needed for Opal™ reagents using single plex stains. Bethyl PathPlex® antibodies are validated to work with Opal™ reagents and need no or minimal adjustments from the suggested optimal dilution. For select PathPlex® antibodies the general staining order has been determined to guide the user during multiplex panel optimization.
Step 2
Using three slides per target, stain slides after first, third or sixth HIER to determine best signal


Step 3
Ordering the antibodies for a Six Plex Panel
Step 4
Assign Fluorophore to Antibodies
Step 5
Order confirmation multiplex IHC



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