Dewax

Tumor section processing and TUNEL staining were performed by HistoWiz (Brooklyn, NY). Briefly, all IHC staining is automated using BOND Rx. Slides were treated with Dewax, a solvent-based solution (Leica Biosystems). Tissue sections were fixed by adding 10% neutral buffered formalin for 15 minutes. Slides were treated with Proteinase K at 1:500 dilution. Tissue sections were re-fixed using 10% neutral buffered formalin. Tissue sections were then treated with the equilibration buffer and incubated for 12 minutes at room temperature (RT). Subsequently the TdT reaction mix was added and incubated for 60 minutes at 37°C. The TUNEL reaction was stopped by adding 2X SSC. Tissue slides were treated with Peroxide Block (3-4% Hydrogen Peroxide) followed by wash buffer. Streptavidin HRP was added and incubated for 30 minutes at RT. DAB was added onto the slides and incubated for 10 minutes. Counter-staining was done by adding hematoxylin. Slides were covered using the Sakura Tissue Tek strainer and cover slips then scanned at 40x using the Leica AT2 scanner.

An automated protocol was used for Panel 2. Slides were heated at 60 °C for 30 min then Dewax (Leica Biosystems, Buffalo Grove, IL) applied to remove any paraffin. Antigen retrieval was performed using ER2 (Leica Biosystems, Buffalo Grove, IL) at 100 °C for 40 min followed by a washing step. Non-specific staining was blocked using Blocking/Ab Diluent (Perkin Elmer, Hopkington, MA) for 5 min, then the first primary antibody was applied, Table 1, followed by a washing step. ImmPRESS™ HRP Anti-Mouse IgG (Vector Laboratories, Burlingame, CA) was applied for 15 min. The slides were washed, and the TSA-dye (Opal 7 color kit, Perkin Elmer, Hopkington, MA) for Position 1 was applied. Slides were then heated using ER1 (Leica Biosystems, Buffalo Grove, IL) at 95 °C for 20 min to strip the primary and secondary antibodies, washed, and blocked again using Blocking/Ab Diluent. The second primary antibody (Position 2) was applied, followed by Opal polymer HRP Ms. + Rb (Perkin Elmer, Hopkington, MA). The corresponding Opal was applied, and the antibodies stripped. The staining process was repeated for positions 3–6. After the last step of antibody striping, the slides were removed from the Bond Rx (Leica Biosystems, Buffalo Grove, IL), and DAPI was applied. After unbound DAPI was washed off, slides were coverslipped using ProLong™ Diamond Antifade Mountant (Life Technologies, Waltham, MA).