Opal 520 reagent pack

Tissue sections were deparaffinized with xylene, rehydrated through graded alcohol and Milli-Q, before incubation in 3.5% formalin for 10 min. Sections were then placed in a cuvette filled with Milli-Q and pH 6 citrate buffer (1:10) and treated with a Decloaking Chamber™ (Biocare Medical, USA) for 10 min at 110 C. Afterward, sections were washed with Milli-Q and TBST Tris (Buffered Saline with Tween 20, Dako) and cooled. Nonspecific binding was blocked by incubation with Antibody Diluent for 10 min. These steps were followed by incubation with the primary antibody of the first marker. After incubation, sections were rinsed in TBST Tris and incubated with the secondary antibody for 20 min, before applying FITC-staining with the Opal™ 520 Reagent Pack (1:100, PerkinElmer) for 10 min. Sections were then washed with TBST Tris and placed in a cuvette filled with AR6 Buffer (PerkinElmer)
List of monoclonal antibodies used. The second marker was applied on the following day. After applying the primary and secondary antibodies of the second marker, sections were Cy5-stained with the TSA™-Plus Cyanine 5 System (1:50, PerkinElmer). Sections were then rinsed in Milli-Q and counterstained with TBST Tris diluted Spectral DAPI (1:12.5, PerkinElmer) for nuclei.
Finally, all tissue sections were mounted with VECTRASHIELD® Har-dSet™ Antifade Mounting Medium (Vector) and coverslipped.