Opal 7 color multiplex ihc kit

Opal™ 7-Color Multiplex IHC Kit (Akoya Biosciences, NEL861001KT) was employed to perform multiplex staining. The protocol was referred to the manufacturer’s construction. FFPE sections were incubated at 65°C for at least 18 h as preprocessing. The slide underwent a serial deparaffinization step and then immersed to quench peroxidase followed by washing. The following steps were repeated for multiple-marker staining. The slides were successively treated for primary antigen retrieval, blocking of unspecific binding, secondary antibody conjugating, and stripping. After completing multiple stainings, the slides were scanned on the PerkinElmer Vectra 3^® Polaris™ platform and imaged on the inForm Advanced Image Analysis software (inForm 2.4.1; Akoya Biosciences, USA) with the DAPI (Akoya Biosciences) filter set. The antibodies and reagents are listed in Table S1.

The formalin-fixed, paraffin-embedded (FFPE) tumor biopsies obtained before (C1D1) and after (C2D8) treatment were analyzed by mIHC staining using the Opal 7-Color Multiplex IHC kit (Akoya Biosciences) as described previously (63 (link)). Briefly, 5 μm FFPE tissue sections were deparaffinized, rehydrated, and refixed with 10% neutral buffered formalin prior to antigen recovery in heated AR9 retrieval buffer (Akoya Biosciences) for 15 minutes. Afterwards, the FFPE sections underwent 6 sequential cycles of staining procedures. Each cycle included blocking, binding of the primary antibody and the corresponding HRP-labeled secondary antibody, and then visualized by a different Opal fluorophore. Each cycle was ended with another heated antigen retrieval process with AR6 retrieval buffer to remove the bound antibody. After the 6-cycle staining procedures, the sections were counterstained with DAPI (Akoya Biosciences) and mounted with Diamond Antifade fluorescence mounting media (Thermo Fisher Scientific). Each single marker with an associated fluorophore staining section served as a reference control in the spectral library for the “spectral unmixing process,” and the unstained slide served as the background control. Each biopsy was used for 2 panels of mIHC staining with the antibodies and corresponding fluorophores listed in Supplemental Table 11.