IHC assay

An IHC assay was conducted to evaluate the expression of RHBDD1 in FFPE NSCLC tissues and matched adjacent tissues using a previously described standard streptavidin–biotin–peroxidase complex method [30]. The samples were cut into 4-μm thick sections, which were deparaffinized in xylene and rehydrated in graded alcohol. Then, they were immersed in 10 mM citrate buffer (pH = 6.0) at 95 °C for 15 min in a microwave oven for antigen retrieval, followed by endogenous peroxidase blocking with 3% hydrogen peroxide for 10 min. Incubation with the primary antibody against RHBDD1 was performed at 4 °C overnight. The sections were rinsed with phosphate-buffered saline (PBS) and incubated with HRP-conjugated secondary antibody at 37 °C for 30 min. After DAB staining and hematoxylin staining, two pathologists independently calculated the IHC staining score of each sample in a blinded manner based on the proportion of the positive-staining cells [31]. The staining intensity scale was scored: 0 (no staining), 1 (weak staining), 2 (moderate staining) or 3 (strong staining). The percentage of stained tumor cells was scored: 1 (1–25%), 2 (26–50%), 3 (51–75%) or 4 (76–100%). The NSCLC samples could then be classified into high RHBDD1 (score ≥ 6) and low RHBDD1 (< 6) groups with the optimal cutoff value being an IHC score of 6 [32].