Multiplex Immunohistochemistry for Tumor-Infiltrating Immune Cells

Hematoxylin/eosin (H/E)-stained slides of formalin-fixed paraffin-embedded (FFPE) tumor blocks were evaluated by an independent pathologist (YMJ). The block with largest area of viable tumors was selected for sectioning at a thickness of 5 μm. The viable tumor parts were marked on H/E slides. Selected FFPE sections were deparaffinized, rehydrated, antigen retrieved, and stained using a customized multiplex fluorescent immunohistochemical (IHC) panel (Opal 7-color manual IHC kit; Akoya, Marlborough, MA, USA) according to the manufacturer’s instructions. The primary antibodies used were CD8 (clone: C8/144B; 1:400) from DAKO (Santa Clara, CA, USA), CD39 (clone: polyclonal; 1:100) from Sigma (St. Louise, MO, USA), CD103 (clone: EPR4166 [2 (link)]; 1:100) from Abcam (Cambridge, UK), and PD-1 (clone: EH33; 1:100) and PD-L1 (clone: E1L3N; 1:200) both from Cell Signaling (Danvers, MA, USA). Spectral 4′,6-diamidino-2-phenylindole was used for nuclear counterstaining. FFPE sections from a tonsillectomy specimen and a PD-L1-high non–small-cell lung cancer specimen were used for the optimization of the staining protocol and as positive controls for PD-1 and PD-L1 staining. FFPE sections from a known CD8 T_RM-rich HCC tumors were included in each staining batch to detect any batch effects as a quality control measure.