Opal 7 color fihc kit

Multiplex immunofluorescence (mIF) staining was performed using Opal 7-Color fIHC Kit (PerkinElmer, Waltham, United States) according to protocols which have been described previously (Parra et al., 2017 (link); Parra et al., 2018 (link)). The embedded tumor tissues that underwent deparaffinization and rehydration were heated at 95°C for 20 min using Tris–EDTA buffer or citrate buffer to retrieve antigen. Next, the slides were incubated with primary antibodies overnight at 4°C. Then, the slides were washed three times with 2-methyl-2H-isothiazol-3-one and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 10 min at room temperature. Next, the slides were incubated at room temperature for 10 min with one of the following Alexa Fluor tyramides included in the Opal 7 kit to detect antibody staining. After BOND Wash Solution washing, the slides were counterstained with DAPI for 5 min to visualize nuclei and then mounted with glycerine. The slides were scanned using the Pannoramic MIDI System (3DHISTECH, Budapest, Hungary). The following primary antibodies are used in this study: CD8 (1:50, Servicebio, Wuhan, China), Ly-6G (1:200, Servicebio, Wuhan, China), and F4/80 (1:200, Servicebio, Wuhan, China).