Immunohistochemical Analysis of SUMO Pathway

Standard immunohistochemical (IHC) staining and the quantitation of the staining were performed as previously described [15 (link)]. Briefly, after de-waxing of the 5μm thick sections using xylene and re-hydration with ethanol, endogenous peroxidase activity was blocked using 3% hydrogen peroxide. This was followed by antigen retrieval, blocking with 10% normal serum, and incubation of the sections with anti-SAE1 (1:500; #ab185552, Abcam, Cambridge, UK), anti-SUMO1 (1:100; #ab32058, Abcam), anti-SUMO2 (1:100; #ab212838, Abcam), and UBC9 (1:100; #ab75854, Abcam) antibodies overnight at 4 °C, followed by goat anti-rabbit IgG (H + L) HRP-conjugated secondary antibody (1:10,000; #65-6120, Thermo Fisher Scientific Inc., Waltham, MA, USA). As chromogenic substrate, Diaminobenzidine (DAB) was used, and the stained sections were counter-stained with Gill’s hematoxylin (Thermo Fisher Scientific, Waltham, MA, USA). The univariate and multivariate analyses were done using the Cox proportional hazards regression model.