Multiplexed Immunofluorescence Staining of FFPE TMAs

Multiplexed fluorescence staining was performed as previously described (39 (link) {Song, 2022 #205)}. In brief, 4-μm FFPE TMAs sections were deparaffinized in xylene and then rehydrated in 100, 90, and 70% alcohol successively. Antigen unmasking was performed with a preheated epitope retrieval solution, endogenous peroxidase was inactivated by incubation in 3% H2O2 for 20 min. Next, the sections were pre-incubated with 10% normal goat serum and then incubated overnight with primary antibodies panel: CD4 antibody (CST, 48274, 1:100), CD8 antibody (CST, 55336, 1:300) and CXCR6 antibody (abcam, ab273116,1:1000). Next, TMA sections were incubated with the corresponding HRP-conjugated goat anti-rabbit second antibodies (ZSBIO, CA) for 10-30 mins at room temperature. The antigenic binding sites were visualized using the OPAL dye: Opal -650 (AKOYA),Opal −520 (AKOYA), Opal- 570 (AKOYA) for each antibody, respectively. Then, TMA sections were counterstained with DAPI (Sigma) for 3-5 mins. Similar to the data analysis of immunohistochemistry, Multiplexed fluorescence staining images were analyzed and quantified by HALO software (Indica Labs, Corrales, NM, USA) as well. For the convenience of downstream comparisons, the CD4⁺CXCR6⁺, CD4⁺CXCR6^-, CD8⁺CXCR6⁺ and CD4⁺CXCR6^- were analyzed, respectively.