Paraffin-embedded tissue blocks were cut into 4 μm sections, dried overnight at 37°C, and then deparaffinized with xylene and rehydrated in a graded ethanol series. Sections were treated with Dako target retrieval solution (Dako, Carpinteria, CA, USA) before antigen retrieval was done by heating at 95°C for 40 min.

Then the sections were cooled to room temperature, and were treated with dilute hydrogen peroxide to block Trichostatin A chemical structure endogenous peroxidase activity. Nonspecific binding was minimized by incubation with Dako protein block (Dako) for 30 min. Rabbit anti-human polyclonal antibodies for metastin (1–54)-Amide (catalogue number: H-048-59, Phoenix Pharmaceuticals, Inc., Burlingame, CA, USA) and GPR54 (375–398) (catalogue number: H-048-61, Phoenix Pharmaceuticals) were applied overnight at 4°C at a dilution of 1:400. On the next day, sections were incubated for 1 hr at room temperature Selonsertib with anti-rabbit IgG conjugated to a horseradish peroxidase (HRP) -labelled polymer (Dako Envision™ + System, Dako), treated with 3,3′-diaminobenzidine tetrahydrochloride (DAB), and counterstained with Mayer’s hematoxylin. As a positive control, human

placental tissue was stained with the anti-metastin and anti-GPR54 antibodies (Figure 1A, 1B). For negative control slides, the primary antibody was substituted with irrelevant rabbit serum. Figure 1 Immunohistochemical staining LCZ696 of non-cancerous pancreatic tissues and pancreatic cancer tissues. (A, B); Immunohistochemical staining of human placental

tissues as a positive control. Tissues were stained with anti-metastin (A) and anti-GPR54 antibody (B). (Original magnification, × 200). (C, D); Non-cancerous and cancerous tissues were stained with anti-metastin and anti-GPR54 antibody. (Original magnification, × 400). Weak positivity of non-cancerous ductal cells for metastin (C) and GPR54 (D). (E, F); Pancreatic cancer tissues were stained with anti-metastin and anti-GPR54 antibody. Heterogeneous strong positive immunostaining of carcinoma cells for metastin (E) and GPR54 (F) are shown. Assessment of metastin and GPR54 expression Five fields (at a × 400 magnification) were randomly chosen to evaluate staining. The intensity of staining in cancer tissues was graded according to a 3-point scale as follows: 0 was weak; 1 was next mild (the same staining intensity as that of non-cancerous pancreatic ducts as an internal control on each slide); and 2 was strong. The percentage of tumor cells showing each staining intensity was estimated to calculate an intensity score ([0 × %weak] + [1 × %mild] + [2 × %strong]) that could range from 0 to 200. A score ≥ 100 was defined as positive staining and a score <100 was defined as negative staining. Then we compared clinicopathological characteristics between patients with positive and negative staining for metastin and GPR54.