Flex 3 3 diaminobenzidine dab substrate working solution and haematoxylin counterstain

Cartilage and synovial tissue from end-stage OA patients was collected during total knee replacement surgery. Cartilage was dissected from the tibial plateau. Cartilage and synovial samples were immersed in 10% formalin for 0.5 mm/hour, embedded in paraffin before cutting 5μm sections and baking onto adhesive glass slides. Deparaffinisation and antigen retrieval procedure was performed using a PT Link machine (Dako, Glostrup, Demark) using FLEX TRS antigen retrieval fluid (Dako). Immunostaining was performed using an Autostainer Link 48 machine using the EnVision FLEX visualisation system (Dako) with anti-human IL-17RA, anti-human IL-17RC antibodies (R&D systems, Abingdon, UK) or universal negative control mouse (Dako) (Supplementary Table 1). Antibody binding was visualized by FLEX 3,3’-diaminobenzidine (DAB) substrate working solution and haematoxylin counterstain (Dako) following the protocols provided by the manufacturer. Antibodies were validated in-house to determine the concentration of antibody needed for positive staining with minimal artefact from the tissue. After staining, slides were dehydrated before mounting using Pertex mounting medium (Histolab, Gothenburg, Sweden). Negative controls are provided in Supplementary Figure 1.