Immunohistochemical Analysis of xCT and 4-HNE

Paraffin-embedded tissue array or xenografts were cut into consecutive 5 μm sections. The sections were rehydrated and antigen retrieval with microwave was performed. Immunohistochemistry staining was conducted using Streptavidin-HRP Rabbit & Mouse DAB Kit (CwBio, Taizhou, China). In brief, endogenous peroxydase enzyme in the sections was blocked using 0.3% H₂O₂ for 10 min, followed by PBS washing for 3 times. Thereafter, the sections were subjected to antigen blocking with 5% goat serum for 30 min, followed by incubation with primary antibody at 37 °C overnight. Primary antibodies were used as follows: xCT (Proteintech, Wuhan, China, Cat#26864-1-AP, 1:100), 4-HNE (R&D, Minneapolis, MN, USA, Cat#MAB3249, 1:300). On the next day, the sections were incubated with biotin-labeled secondary antibodies, streptavidin-HRP, and DAB sequentially. Then, sections were subjected to hematoxylin couterstaining for 1 min. The immunostained sections were captured with the 3D HISTECH digital Scanner (Pannoramic MIDI, Budapest, Hungary) at 400 × magnification. The xCT and 4-HNE expression levels were analyzed using the H-score method by two pathologists as previously described [11 (link)]. H-score = 1 × (% cells 1+) + 2 × (% cells 2+) + 3 × (% cells 3+).