Pd 1 315 m 96

Sequential dual immunofluorescence (IF) was performed on paraffin-embedded tissues mounted onto slides. Tissue sections were labeled for the following antigens: dual CD4/CD8 (NCL-L-CD4-368 and NCL-L-CD8-4B11, Leica Microsystems Inc.), STAT1 (9175, Cell Signaling Technology), CD163 (163 M-18, Cell Marque), PDL-1 (13,684, Cell Signaling Technology), and PD-1 (315 M-96, Cell Marque). This assay was carried out on the Leica Bond Rx fully automated slide staining system (Leica Biosystems) using the Bond Research Detection kit (DS9455). Slides were dewaxed in Bond Dewax solution (AR9222) and hydrated in Bond Wash solution (AR9590). Heat-induced antigen retrieval was performed at 100ºC in Bond-Epitope Retrieval solution 1 pH-6.0 (AR9961) for 20 or 10 min. After pretreatment, tissues were blocked, and primary antibodies were diluted. Ready-to-use secondary antibodies, Leica’s Novolink Post Primary and/or Novolink Polymer (RE7260-CE) were used, followed by either TSA Cy5 (SAT705A001EA, Akoya Biosciences) or TSA Cy3 (SAT704A001EA, Akoya Biosciences) to visualize the target of interest. Nuclei were stained with Hoechst 33,258 (Invitrogen). The stained slides were mounted with ProLong Gold antifade reagent (P36930, Life Technologies). Positive controls were included for each assay.