Deparaffinized sections were pre-treated to retrieve antigens before detection with a rabbit polyclonal anti-NRF2 (Invitrogen) at a 1:200 dilution, and a rabbit polyclonal anti-ACSS2 (Sigma-Aldrich) at a 1:25 dilution. Cytokeratin 5 immunohistochemistry with anti-CK5 (Invitrogen) was used to validate the antigenicity of human tissue sections.
Human ESCC formalin-fixed paraffin-embedded tissue sections were decoded without patient information. IHC staining was scored based on a positive staining area and staining intensity. Numeric scores were provided for staining area and staining intensity: 3 for high intensity or a high percentage of positive staining (>75% cancer cells); 2 for moderate staining or moderate percentage (50–75%); 1 for low staining intensity or low percentage of positive staining (25–50%); and 0 for no staining. A combined numerical score for each antigen was generated by adding the numerical score of the percentage of positive staining to that of the staining intensity.
Human ESCC formalin-fixed paraffin-embedded tissue sections were decoded without patient information. IHC staining was scored based on a positive staining area and staining intensity. Numeric scores were provided for staining area and staining intensity: 3 for high intensity or a high percentage of positive staining (>75% cancer cells); 2 for moderate staining or moderate percentage (50–75%); 1 for low staining intensity or low percentage of positive staining (25–50%); and 0 for no staining. A combined numerical score for each antigen was generated by adding the numerical score of the percentage of positive staining to that of the staining intensity.