Immunohistochemical Staining of CST3 Protein

An automatic staining machine (BenchMark XT; Ventana Medical Systems, Tucson, AZ, USA) was used for immunohistochemical staining using the XT ultraView (UV) 3, 3′-diaminobenzidine (DAB) detection kit (Ventana Medical Systems). Tissue sections (4 μm) on slides were deparaffinized, hydrated, and heated to induce antigen retrieval according to a previously described method [40 (link)]. After inactivating endogenous peroxidase activity using a UV inhibitor, the sections were incubated with a rabbit monoclonal anti-CST3 antibody (ab109508; Abcam, Cambridge, MA, USA) or a control antibody (1:5000) in a blocking solution at 37 °C for 30 min. Sections were rinsed and then incubated with UV horseradish peroxidase (HRP) at 37 °C for 8 min. Staining signals were developed using UV DAB and hydrogen peroxide at 37 °C for 8 min. Slides were finally incubated with UV copper for 4 min to enhance signal intensity, counterstained with hematoxylin (Vector Laboratories, Burlingame, CA, USA), and photographed using TissueFaxs software on an automated acquisition system (TissueGnostics, Vienna, Austria).