Novolink max polymer kit

For immunofluorescence (IF) staining, Opal stain kit (PerkinElmer, Waltham, MA, USA) was employed. After cell fixation, antigen retrieval was performed with citrate buffer (pH 6.0) in a microwave oven. The slides were then washed, blocked, and incubated with β-TrCP primary antibody at 4 °C overnight followed by incubation with secondary antibody polymer HRP for 10 min and subsequently with Opal fluorophore for 10 min at room temperature. To further stain with anti-NRF2 antibody, the slides were again placed in citrate buffer (pH 6.0) and heated in a microwave oven. After Opal staining process, DAPI was applied for nuclei staining.
For immunochemistry (IHC) staining, Novolink Max Polymer kit (Leica Biosystems, Wetzlar, Germany) was employed. All slides were dewaxed with xylene/ethanol, and then antigen retrieval was performed with TRS buffer (pH 6.0) in a microwave oven. After blocking, the slides were reacted with primary antibodies. The peroxidase activity was visualized with diaminobenzidine tetrahydroxychloride (DAB) solution. The sections were counter stained with hematoxylin. Dark brown staining was considered positive. The antibody conditions are described in Table S2.

Tumor sections derived from syngeneic mouse models were prepared by the Pathology Core Facility, Institute of Biomedical Sciences (Academia Sinica, Taiwan). IHC staining was performed with a Novolink Max Polymer kit (Leica Biosystems, Wetzlar, Germany) according to the manufacturer’s instructions. Briefly, all slides were dewaxed with xylene/ethanol, and antigen retrieval was performed by boiling in TRS buffer for 10 min. Protein Block was used to cover the slides for 10 min followed by incubation with primary antibodies overnight. The polymer was used for polymerization. The peroxidase activity was visualized with diaminobenzidine tetrahydroxychloride (DAB) solution. The sections were counterstained with hematoxylin (Sigma-Aldrich).

Primary lung cancer tissue specimens and microarrays were obtained from the Tissue Bank of National Cheng Kung University Hospital. A total of 119 tumor tissues and 62 adjacent non-tumor tissues were analyzed. Tumor sections derived from xenograft models were prepared by Pathology Core Facility, Institute of Biomedical Sciences (Academia Sinica, Taiwan). IHC staining was performed with Novolink Max Polymer kit (Leica Biosystems, Wetzlar, Germany). Slides were dewaxed with xylene/ethanol and antigen retrieval was performed with TRS buffer in a microwave oven. After blocking, the slides were incubated with primary antibodies. The peroxidase activity was visualized with diaminobenzidine tetrahydroxychloride (DAB) solution. The sections were counter stained with hematoxylin.