Diaminobenzidine tetrahydrochloride

An antibody cross-reactivity tissue array, MNO341 (US Biomax, Rockville, MD, USA), which contained 33 tissue types mostly from surgical resection, was used to examine the tissue distribution of each peptide. The tissue preparations were deparaffinized, rehydrated, and autoclaved in pH 6.0 citric acid buffer for antigen retrieval. The preparations were blocked using an endogenous Avidin Biotin Blocking Kit (Nichirei Bioscience, Tokyo, Japan) and then incubated overnight at 4 ℃ with either anti-SBSN_HUMAN[225–237] IgG, anti-SBSN_HUMAN[243–259] IgG, anti-SBSN_HUMAN[279–295] IgG or control non-immune IgG purified from preimmune serum (1:1000 dilution). After washing in PBS, the samples were incubated with a biotinylated anti-rabbit IgG antibody (1:3000, Abcam) for 60 min at room temperature. Antibody binding was visualized by the avidin–biotin-complex peroxidase method using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA) with diaminobenzidine tetrahydrochloride (Nacalai Tesque). The tissue preparations were stained with hematoxylin (Muto Pure Chemicals CO, Tokyo, Japan)^{60 (link)}.