Histofine simple stain max po r or m kits

Paraffin‐embedded tissue sections (3.5 µm) and cells cultured on chamber slides were immunostained using Histofine Simple Stain Max PO (R) or (M) kits (Nichirei Biosciences Inc.). Endogenous peroxidase activity was blocked by incubating the sections with 0.3% hydrogen peroxide in methanol for 30 minutes. The slides were then incubated for 20 hours at 4°C in PBS containing 1% BSA (Sigma‐Aldrich Co.), 1:1000‐diluted rabbit anti‐chromogranin antibody (A0430; Dako), 1:300 rabbit anti‐synaptophysin antibody (A0010; Dako) , 1:200 diluted mouse anti‐Ki‐67 antibody (M7240; Dako)12 or 1:100 mouse anti‐INSM1 (sc‐271408; Santa Cruz Biotechnology). Bound antibodies were detected using Simple Stain Max PO (R) or (M) with diaminobenzidine‐tetrahydrochloride (DAB) as the substrate, and visualized by counterstaining with Mayer’s hematoxylin. Negative controls were not stained with the primary antibodies. When the immunoreactivity for CgA or synaptophysin was found to be positive in more than 50% of the tumor cells, a diagnosis of NEC was made in accordance with guidelines established in a previous report.13