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 Supplementary Figure 1
Autostainer link 48 machine
Manufactured by Agilent Technologies
Sourced in United Kingdom, Denmark
The Autostainer Link 48 is an automated slide staining system designed for immunohistochemistry and in situ hybridization applications. It is capable of processing up to 48 microscope slides simultaneously, providing consistent and reliable staining results.
Automatically generated - may contain errors
Lab products found in correlation
Pt link machine, by Agilent Technologies (1 mentions)
Envision flex visualisation system, by Agilent Technologies (1 mentions)
Flex 3 3 diaminobenzidine dab substrate working solution and haematoxylin counterstain, by Agilent Technologies (1 mentions)
Envision flex kit, by Agilent Technologies (1 mentions)
Ab260032, by Abcam (1 mentions)
2 protocols using autostainer link 48 machine
1
Immunohistochemical Analysis of IL-17 Receptors in OA
2
Immunohistochemistry for ADORA2A and TNFRSF18
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Rabbit monoclonal antibodies for ADORA2A (ab260032) and TNFRSF18 (ab223841) were purchased from Abcam (USA). After slicing into 4-µm sections, all tissues were deparaffinized and treated with ethylenediamine tetraacetic acid (EDTA) (pH 9.0) for antigen retrieval in a microwave for 20 min. We used an Autostainer Link 48 machine (Dako, Denmark A/S, Denmark) for the staining. Subsequently, primary antibodies for ADORA2A (rabbit monoclonal, 1:100 dilution) and TNFRSF18 (rabbit monoclonal, 1:200 dilution) were added to the sections, while phosphate buffered saline (PBS) buffer was used as a blank control instead of the antibody. An EnVision Flex Kit (Dako, Denmark A/S, Denmark) was used as the second antibody. Two senior pathologists examined all cases to validate the initial scores. The percentage of positively stained cells and staining scores were used to assess the IHC results according to the methodology described in previous articles (7 (link),8 (link)). The Gene-Score = (percentage of cells of weak intensity ×1) + (percentage of cells of moderate intensity ×2) + percentage of cells of strong intensity ×3).
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