All paraffin-embedded tissues were sectioned at 4 μm, and the workflow was carried out as the manual of EnVision™ Detection Systems Peroxidase/DAB (Dako, Denmark, #K5007). Heat-induced technique was used for the epitope retrieval, followed by primary and secondary antibodies incubation. DAB developer and hematoxylin staining were applied in turn. PBS instead of the primary antibodies were negative controls [14 (link)]. Ventana ALK assay (Predilute D5F3 antibody) were employed to detect the ALK rearrangement on Ventana Benchmark XT platform using the in-house validated protocol [20 (link)].
Two pathologists evaluated the IHC staining blindly without any knowledge of the patients’ clinical information in a semiquantitative method. In each slide, ten areas were randomly selected under light microscopy, and scored for both of the quantity and intensity of positively stained cells. Quantity was scored as 0 for no staining; 1 for < 20% cells stained; 2 for 20–50% cells stained; and 3 for > 50% cells stained; whereas intensity was referred to as 0 for no appreciable staining; 1 for barely detectable staining; 2 for readily appreciable brown staining and 3 for dark brown staining. Quantity scores multiplied by intensities were the total scores, and 0–2 scores were negative, while positive for the others [14 (link)].
Two pathologists evaluated the IHC staining blindly without any knowledge of the patients’ clinical information in a semiquantitative method. In each slide, ten areas were randomly selected under light microscopy, and scored for both of the quantity and intensity of positively stained cells. Quantity was scored as 0 for no staining; 1 for < 20% cells stained; 2 for 20–50% cells stained; and 3 for > 50% cells stained; whereas intensity was referred to as 0 for no appreciable staining; 1 for barely detectable staining; 2 for readily appreciable brown staining and 3 for dark brown staining. Quantity scores multiplied by intensities were the total scores, and 0–2 scores were negative, while positive for the others [14 (link)].
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