IHC Quantification of SERPINE2 and CA9

IHC was performed as previously described [26 (link)], and the primary antibodies for IHC staining included rabbit anti-SERPINE2 (AB155549, Abcam, USA) and anti-CA9 (AB243660, Abcam, USA). The IHC results were scored as H-score, including semi-quantitative grades by “IHC Profiler (Macro)” of ImageJ (version 1.53a): negative, low-positive, positive, and high-positive. The H-score = 0 * the percentage of negative cells + 1 * the percentage of low positive cells + 2 * the percentage of positive cells + 3 × the percentage of high-positive cells, which ranges from 0 to 300 [27 (link)].