Multiplex immunohistochemistry staining and imaging.
4 μm Formalin-fixed, paraffin-embedded tissue sections were used for imaging. Slides were heated for at least 1 hour in a dry oven at 60°C. The slides were dewaxed with Leica Bond Dewax solution (Leica Biosystems), and antigen retrieval was performed with Bond Epitope Retrieval 2 (Leica Biosystems) for 30 minutes. Primary antibody incubation was performed for 30 minutes after blocking with 1× antibody diluent/block solution (Akoya Bioscience) followed by OPAL polymer HRP incubation for 10 minutes. Primary antibodies used included CD4 (EPR6855), CD20 (L26), FoxP3 (236A/E7), and PD-1 [EPR4877(2)] (all from abcam), CXCR5 (D6L3C, Cell Signaling Technology), and CXCL13 (R&D Systems). OPAL Polymer HRP anti-mouse were used for CD20, CXCR5, and FoxP3; anti-rabbit antibodies were used for CD4 and PD-1; and anti-goat were used for CXCL13. Visualization of the antigen was performed using tyramide signal amplification (Akoya Biosciences) for 10 minutes, and, to remove bound antibodies, slides antigen retrieval was performed with Bond Epitope Retrieval 1 (Leica Biosystems) for 20 minutes. The process from blocking to antigen retrieval was repeated for every antibody used. For counterstaining, nuclei were stained with DAPI (Thermo Fisher Scientific). Stained slides were imaged by using the Vectra Polaris Automated Quantitative Pathology Imaging System (Akoya Biosciences). Representative images for training were selected by the Penochart (Akoya Biosciences), and the negative and positive of each marker were trained using the inForm Image Analysis software (Akoya Bioscience) to validate the markers. Each single cell was segmented based on DAPI, and phenotyping was performed according to the expression and intensity of each marker. We designated CXCL13