Opal 7 color fluorescence immunohistochemistry kit

Multiplex immunofluorescence staining was performed with the Opal 7-color fluorescence immunohistochemistry kit (Akoya Biosciences, Marlborough, MA), according to the manufacturer’s protocol, except for the repetitive heat-mediated antibody-stripping treatments. In short, after deparaffinization, rehydration, and blocking endogenous peroxidase, HIER was performed for 5 min. at 95C in Tris-EDTA pH 9.0 buffer in a Lab Vision PT Module (Thermo Fisher Scientific). Sections were washed in TBST and blocked with blocking/antibody diluent for 10 min, before being incubated with primary antibody for 60 min. Then, sections were incubated with polymer HRP Ms + Rb for 10 min, followed by incubation with an Opal fluorophore (Opal480, Opal520, Opal570, Opal620, Opal690, or Opal780) for 10 min. Bound primary and secondary antibodies were then eluted with HIER treatment (as aforementioned) or with β-mercaptoethanol-containing stripping buffer (defined as above) for 30 min in a water bath at 50C. After washing in cold running tap water, demi water, and TBST, the process of staining and antibody removal was repeated using a different Opal fluorophore. Finally, after staining with the sixth Opal fluorophore, tissue specimens were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min and mounted in ProLong Diamond Antifade Mountant (ThermoFisher Scientific).