Antibody clone d5f3 3633

Expression of ALK protein and scoring were described previously [11 (link)]. Briefly, antibody clone D5F3 #3633 (Cell Signaling, Denver, MA, USA) was used at dilution 1:250 and antigen retrieval was performed using EnVision FLEX HIGH pH solution (DAKO) for 30 min. at 99 °C. ALK scoring was applied as the following: 0 (negative) for 0–10%, 1 + for > 10–50%, 2 + for > 50–100% of positive tumor cells. Whole preparations were scanned in Hamamatsu NanoZoomer 2.0 RS scanner at original magnification 40 ×.

Immunohistochemical reactions were performed on 4 μm thick FFPE preparations. Expression of TFAP2B protein was detected using rabbit anti-AP-2β antibody (sc-8976, dilution 1:500; Santa Cruz Biotechnology, Santa Cruz, CA). Antigen retrieval was performed at pH 6.0 for 30 min. at 99 °C. Expression of ALK protein was detected using antibody clone D5F3 #3633 (Cell Signalling, Denver, MA, USA) at dilution 1:250 and antigen retrieval was performed using EnVision FLEX HIGH pH solution (DAKO) for 30 min. at 99 °C. Expression of OLIG2 was detected using antibody EP112 (Cell Marque) at concentration 1.92 μl/ml and antigen retrieval was performed in CC1 buffer using Ventana BenchMark ULTRA IHC/ISH system (Roche).
TFAP2B and ALK scoring were as the following: negative for < 10%, intermediate for 10–50% and positive for > 50–100% of tumour cells. OLIG2 was considered positive when > 10% of tumour cells displayed positive nuclear reaction.
Whole preparations were scanned in Hamamatsu NanoZoomer 2.0 RS scanner at original magnification 40x.