Automated acquisition system

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).

The use of human tissues or tumour specimen samples was approved by the IRB of Taipei Veterans General Hospital. For immunohistochemical staining, paraffin-embedded sections were deparaffinized and rehydrated, with antigen being retrieved by placing sections in Declere working solution (Cell Marque, Austin, TX) in 95 °C water for 30 min. Endogenous peroxidase activity was blocked by 3% hydrogen peroxide. Residual enzymatic activity was removed by washes in PBS, and nonspecific staining was blocked with Ultra V Block for 5 min (Thermo Fisher Scientific, Fremont, CA). Then, the sections were reacted with primary antibodies (pS727STAT3 (catalogue No. 9136; 1:25) and Col17a1 (catalogue No. ab28440; 1:50)), followed by corresponding biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA), and then these sections were treated with streptavidin-peroxidase (LSAB Kit; Dako, Carpinteria, CA), followed by diaminobenzidine staining. Counterstaining was performed with Mayer's haematoxylin and photographed with Zeiss AxioImager Z1 microscope system (Wetzlar, Germany) and an automated acquisition system (TissueGnostics, Vienna, Austria). Pictures were acquired using the Tissue-Faxs software (TissueGnostics). The percentage of positively stained cells was determined using the HistoQuest software (TissueGnostics).