Opal fluorophore conjugated tyramide signal amplification reagent

Using an Opal 7-color Kit (Akoya Biosciences, Marlborough, MA, USA), multiplex immunofluorescence staining was conducted on TMA sections according to the manufacturer’s protocol and as previously described by Yeong et al. (24 (link)) for simultaneous detection of CD68, myeloperoxidase (MPO), citrullinated histone H3, and DAPI. The TMA sections were baked at 65°C for 2 h and subjected to deparaffinization, rehydration, and heat-induced epitope retrieval. The sections were then incubated with a primary antibody for 1 h at room temperature, followed by incubation with an anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (Akoya Biosciences). We incubated the slides with an opal fluorophore-conjugated tyramide signal amplification reagent (Akoya Biosciences). Heat-induced epitope retrieval was performed to remove the bound antibody complexes. The same procedure was repeated until all targets were detected, and the samples were labeled with DAPI (Akoya Biosciences). The following antibodies were used: anti-CD68 (D4B9C; Cell Signaling Technology, Danvers, MA, USA)/Opal 520, anti-citrullinated histone H3/Opal 620 (ab5103; Abcam, Cambridge, UK), MPO (E1E7I; Cell Signaling Technology, Danvers, MA, USA)/Opal 690. Finally, the sections were mounted using a hard-set medium.

The mIHC/IF staining was performed with an Opal Polaris 7-Color Multiplex IHC kit from Akoya Biosciences. Briefly, each FFPE tissue section was baked at 65 °C for 1 h. After deparaffinization, rehydration, and microwave antigen repair, the slides were blocked (Akoya Biosciences, USA), and incubated with primary antibodies against CD3 (D7A6E, CST, 85061 T, 1:200), CD57 (HNK-1, CST, 72031 S, 1:200), and CD8 (EPR22483-288, Abcam, ab245118, 1:500), respectively, followed by incubation with Opal Polymer HRP Ms+Rb (Akoya Biosciences, USA). The slides were then incubated with Opal Fluorophore-conjugated tyramide signal amplification reagent (Akoya Biosciences, USA). After signal amplification, microwave antigen repair was performed to remove the detected antibodies. This process was repeated using another Opal Fluorophore. The above steps were repeated until the slides were labeled with all antibodies and DAPI. Finally, the slides were sealed with an anti-fluorescence quencher. Visualization of slides was done using Vectra Polaris Quantitative Pathology Imaging Systems (Akoya Biosciences, USA); analysis and scoring were performed using inForm software (Akoya Biosciences, USA). The CD57⁺CD8⁺ T cells/T cell ratio, CD57⁺CD8⁺ T cells/ CD8⁺ T cells ratio, and total CD8⁺ T cells/T cells ratio were calculated the same as flow cytometry.