Pt link machine

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.

We performed immunohistochemical (IHC) staining for HER3 as previously described [34 (link), 35 (link)]. Briefly, sections of each sample were deparaffinized and antigen retrieval was performed at high pH (PT Link machine, Dako). The sections were then stained with a rabbit monoclonal antibody against HER3/ErbB3 (1:59 dilution; clone D22C5, Cell Signaling Technology Inc., Danvers, MA, USA) using the Dako autostainer Link48 (Dako, CA, USA) and EnVision Flex Mini Kit (Dako), according to the manufacturer’s instructions. Hematoxylin was used as a nuclear counterstain. HER3-high was defined as an IHC score of 3 + or 2 + , and HER3-low/zero was defined as an IHC score of 1 + or 0 in line with the HER2 testing guidelines for gastroesophageal cancer [36 (link)]. The H-score (range, 0–300) was calculated using the following formula: 3X + 2Y + Z, where X, Y, and Z are the percentages of tumor cells showing strong, moderate, and weak staining intensities, respectively [37 (link)] (Additional file 1: Table S1A–D). The specificity of this HER3 IHC staining method was validated by confirming no signal detected with an isotype control antibody. Both positive and negative cell line control slides were also incorporated into HER3 staining to verify the specificity at every batch of staining.

Mice were sacrificed at 5 weeks, 3 months, or 5 months post-injection. Livers were removed and fixed overnight in 4% formalin, then embedded in paraffin, and cut into 5-μm sections. Immunohistochemistry was performed using the EnVision FLEX Mini Kit (DAKO). Antigen retrieval was done in a PT Link machine (DAKO). The primary antibodies used for immunohistochemistry are rabbit polyclonal anti-FAH (ThermoFisher Scientific, PA5-42049, 1:100), incubated for 120 min, and rabbit polyclonal anti-alpha 1 fetoprotein (Abcam, ab46799, 1:500) and rabbit polyclonal anti-glypican 3 (Abcam, ab186872, 1:800), incubated overnight. Secondary antibody polyclonal goat anti-rabbit-HRP (DAKO, P0448) was incubated for 30 min. Visualization was done with EnVision FLEX DAB+ Chromogen System (DAKO, GV825). After hematoxylin counterstaining for 5 min, slides were mounted and scanned with a Pannoramic Digital Slide Scanner (3D Histech).