Multiplex Immunofluorescence Staining Protocol

Tissue sections (4 µm) prepared from formalin-fixed paraffin embedded tissue blocks were baked at 58 °C for 1 hour. If tissue detachment (commonly exhibited as lifting edges of sections) was observed following a staining procedure, a longer baking time was applied to revised protocols to enhance tissue attachment. After baking, tissue sections were deparaffinized with three treatments of xylene for 5 min each, followed by three rinses with 100% ethanol for 2 min each. Sections were rehydrated by rinsing in 95% ethanol, 70% ethanol, and water for 5 min each. Initial antigen retrieval was performed in a decloaking chamber (Biocare Medical LLC, Concord, CA) with 10mM citrate buffer (pH 6.0) for 45 min at 125 °C at 15psi.
An Opal 7-color manual IHC kit (Akoya Bioscience) was used for manual mIF staining with the same antibodies and Opal dye dilutions used for automated staining (Table 1). Tissue sections were incubated with the first primary antibody diluted in Antibody Diluent/Blocking solution provided in the kit followed by washing in TBST buffer and then incubated with the secondary-HRP conjugate. After another wash, slides were incubated with the appropriate Opal fluorophore. The HIER step for removing the primary-secondary-HRP complex between staining rounds was achieved by placing the slides in a plastic coplin jar filled with antigen retrieval buffer (AR6 provided in the kit), placing the jar in a 1100-watt microwave, and heating for 45 min at 100% power followed by 14 min at 20% power. After the slides cooled to room temperature they were washed and then were ready for the next round of staining with the next primary antibody. After the last round of antibody staining, slides were counterstained with Spectra DAPI, then mounted with Fluoromount-G Mounting Medium (Thermo Fisher Scientific).