Figure 1. Multiplex fluorescent immunohistochemistry allows phenotyping of 7 unique cell types in the colorectal liver metastasis tumor microenvironment.

(A) A composite image was created incorporating all of the fluorophores present in a single core after multispectral imaging. (B–F) After construction of a single-stained library, spectral unmixing allows imaging of single fluorophores representing (B) pancytokeratin, (C) CD3, (D) CD8, (E) FoxP3, (F) CD163, and (G) DAPI. (H) A single unstained slide processed similarly to the tissue microarray (TMA) but without addition of primary antibodies or tyramide signal amplification (TSA) allows for measurement and subtraction of autofluorescence.