Nucleus pulposus tissues were obtained from case and control groups (34 cases and 21 controls). Immunohistochemical assay was performed using a standard protocol as previously reported.[27 (link)–29 (link)] The sections were treated with 1/200 IL-1α antibody (ab7632, Cambridge, MA, USA), 1/200 IL-1β antibody (#2022, Cell Signaling, Danvers, MA, USA), 1/400 IL-1RN antibody (ab123235, Abcam, Cambridge, MA, USA) overnight at 4 °C and incubated with 1/400 secondary biotinylated goat anti-rabbit antibody (Vector Laboratories, Burlingame, CA, USA) for 30 min, followed by treatment with VECTASTAIN Elite ABC Kit (Vector Laboratories, Burlingame, CA, USA). Based on stained slides, the number of positive cells was manually counted using Olympus BX43 upright microscope.