Biotinylated goat anti mouse antibody

Free floating sections were quenched in 0.3% H₂O₂ for 25 min, then blocked in 5% goat serum 90 min. They were then incubated with primary antibody mouse anti-parvalbumin (Sigma Aldrich) for 2 days followed by a biotinylated goat anti-mouse antibody (Sigma Aldrich) for 90 min. Following this, solutions A and B from the standard Vectastain ABC kit (Vector Laboratories) 1:500 for 1 h. Staining was developed using a DAB (3,3′-Diaminobenzidine tetrahydrochloride hydrate; Sigma Aldrich) solution. The sections were then mounted onto slides. After air drying, slides were dehydrated in increasing concentration of alcohol, cleared with Hemo-De and coverslipped with Fisher Chemical Permount^™ Mounting Medium.

Thirty formalin-fixed paraffin embedded gastric cancer tissue samples that were used for the above macromolecule analysis were also used for immunohistochemistry (IHC). Two micrometer sections were cut the day before use and stained according to standard protocols. Two strategies were used to maximize the IHC signal: antigen retrieval in citrate buffer, and signal amplification with biotinylated tyramide, as described previously (Yoon et al., 2011b (link)). The sections were incubated overnight at 4 °C with HER-2 antibody (1/100; Sigma, St. Louis, MO, USA). Detection was carried out using biotinylated goat anti-mouse antibody (Sigma), followed by incubation with a peroxidase-linked avidin–biotin complex. Diaminobenzidine was used as the chromogen, and the slides were counterstained with Mayer's hematoxylin. Two experienced pathologists assessed HER-2 immunostaining using the HercepTest (Dako, Carpentaria, CA, USA). Modified HER-2 scoring criteria for gastric cancer were used; 0, no staining or <10% tumor cells with membrane staining; 1+, >10% tumor cells with faint staining in partial membrane; 2+, >10% tumor cells with weak to moderate staining in partial membrane; 3+, >10% tumor cells with strong staining in partial membrane (Hofmann et al., 2008 (link)). Themanufacturer provided the positive and the negative control.