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.