ALK IHC Protocol for NSCLC

The presence of ALK overexpression was assessed by immunohistochemistry (IHC) staining using 4-μm-thick sections. Ganglion cells present in sections of the appendix were used as positive controls, and in negative controls, the primary antibody was omitted. Immunohistochemical reactions were performed at Ventana Benchmark XT using the Ventana ALK (D5F3) CDx assay (Ventana, Tucson, AZ, clone 790–4796) according to the manufacturer. In brief, slides of the NSCLC tumor were subjected to deparaffinization using EZ Prep (Ventana, Tucson, AZ) and antigen retrieval was performed using Cell Conditioning 1 (Ventana, Tucson, AZ). Tissue sections were then incubated with anti-ALK antibody (clone D5F3, Ventana, Tucson, AZ) for 20 min. The OptiView DAB IHC Detection Kit (Ventana, Tucson, AZ) and OptiView Amplification Kit (Ventana, Tucson, AZ) were used according to the manufacturer’s recommendations for the visualization of the bound primary antibody. The ALK stain was considered positive if at least one cell presented strong dark brown cytoplasmic staining as stated in the kit’s manual as previously described [20 (link)].