Multiplex Immunohistochemistry for Tumor Microenvironment

mIHC was performed using the Opal 7-color IHC kit (PerkinElmer) as previously described.25 (link) Briefly, FFPE sections (4 µm thick) were dewaxed and rehydrated. Antigen retrieval was performed in citrate buffer (PH=6.0), and endogenous peroxidase was quenched using 3% H₂O₂ for 15 min. The slides were blocked with 2% bovine serum albumin for 15 min and then incubated with primary antibodies of two panels overnight, including CD3, CD4, CD20, Foxp3 and PD-1; CD8, CD56, CD68, CD163 and PD-L1 (online supplemental table 1). Then, slides were incubated with corresponding HRP-conjugated secondary antibodies and fluorescent dyes with the following order: Opal540, Opal570, Opal620, Opal650 and Opal690 (PerkinElmer). Nuclei was stained with DAPI (PerkinElmer). Subsequently, the slides were scanned and imaged using the Mantra Quantitative Pathology Workstation (PerkinElmer, Waltham, Massachusetts, USA). Images were obtained for the following analysis using the inForm Advanced Image Analysis software (V.2.4.2, PerkinElmer). Multispectral images were unmixed using spectral libraries obtained from images of single-stained slides for each marker. For subregions with multiple TLSs, the densities and frequencies of each marker were averaged to represent the cellular composition.