Paraffin-embedded clinical NSCLC samples and metastatic lung tumors in mouse models were subjected to IHC staining as previously described [21 (link)]. For the detection of FXR, IL-6, IL6ST and p-STAT3, the following primary antibodies were used: anti-bile acid receptor NR1H4 (1:100; cat. no. ab187735; Abcam, Cambridge, UK), anti-IL-6 (1:100; cat. no. ab9324; Abcam), anti-CD130 (gp130) (1:100; cat. no. ab227058; Abcam) and anti-Phospho-STAT3 (Tyr705) (1:200; cat. no. 9145s; Cell Signaling Technology, Inc., Beverly, MA). Concentration-matched non-specific mouse or rabbit IgG served as isotype controls. The IHC staining results were scored independently and blindly by two skilled pathologists, and a final consensus was reached. The staining intensity of tumor cells was scored as negative (0), weak (1), medium (2), and strong (3), respectively. The percentage of positive cells was scored as follows: 0% (0), 1%–25% (1), 26%–50% (2), 51%–75% (3), and 76%–100% (4), respectively. The final IHC scores of human FXR, IL-6, IL6ST and p-STAT3 were obtained by multiplying the staining intensity score with the positive-cell percentage score, and stratified as follows: Low, score < 6 or high, score ≥ 6, in line with our previous studies [18 (link), 19 (link)].
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