Dual-Immunostaining for SIRT1 and ISG15

Dual-immunohistochemical staining for SIRT1 and ISG15 was performed using a Ventana BenchMark ULTRA system (Roche Korea Diagnostics Ltd., Seoul, Korea). Antigen retrieval was performed for 64 min using pH 9.0 CC1 antigen retrieval solution (Roche Korea Diagnostics Ltd.). The specimens were incubated with primary antibodies (anti-SIRT1 (sc-15404), 1:100, Santa Cruz; and anti-ISG15 (HPA004627), 1:100, Atlas Antibodies) for 90 min, and developed with an OptiView DAB IHC Detection Kit (Roche Korea Diagnostics Ltd.) for SIRT1 and with an UltraView Universal Alkaline Phosphatase Red Detection Kit (Roche Korea Diagnostics Ltd.) for ISG15. For quantification, the combined expression of SIRT1 and ISG15 (SIRT1/ISG15) and the individual expression of SIRT1 or ISG15 were assessed. The combined expression of SIRT1 and ISG15 (SIRT1/ISG15) and the individual expression of SIRT1 or ISG15 were scored by summing the staining intensity score (0; no staining, 1; weak staining, 2; intermediate staining, 3; strong staining) and the area score (0; 0%, 1; 1%, 2; 2–10%, 3; 11–33%, 4; 34–66%, 5; 67–100%)^{40 (link)}. The final score was assessed by summing the scores from the two tissue microarray cores, resulting in a final immunohistochemical staining score ranging from zero to sixteen^{41 (link)}.