Biotinylated goat anti rabbit immunoglobulin g igg

Immunohistochemistry (IHC) was performed using the avidin-biotin-peroxidase complex method with all breast carcinoma samples. All sections were deparaffinised in xylenes and dehydrated through a gradient concentration of alcohol before endogenous peroxidase activity was blocked using 0.5% H₂O₂ in methanol for 10 min. After non-specific binding was blocked, the slides were incubated with mouse monoclonal antibodies for ER (1D5) (1:50) and PR (PgR 636) (1:400) in phosphate-buffered saline (PBS) at 4°C overnight in a humidified container respectively. Biotinylated goat anti-rabbit immunoglobulin G (IgG) (1:400; Sigma-Aldrich, St Louis, MO, USA) was incubated with the sections for 1 h at room temperature and detected using a streptavidin-peroxidase complex. The brown color indicative of peroxidase activity was developed by incubation with 0.1% 3,3′- diaminobenzidine (Sigma-Aldrich) in PBS with 0.05% H₂O₂ for 5 min at room temperature. Polyclonal HER2 antibody in the Herceptin kit (HercepTest, DAKO, Denmark) was used according to the manufacturer's instructions. Positive controls of known positive breast cancer tissues and negative controls with primary antibody replaced with TBS were run with the patient slides in each run of IHC.

Immunohistochemistry (IHC) was performed using the avidin-biotin-peroxidase complex method on all breast carcinoma samples. All sections were deparaffinised in xylenes and dehydrated through a gradient concentration of alcohol before endogenous peroxidase activity was blocked using 0.5% H₂O₂ in methanol for 10 minutes. After nonspecific binding was blocked, the slides were incubated with NDRG2 antibody (1:200; Abnova, Taipei, Taiwan) or GLUT1 antibody (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA) in phosphate-buffered saline (PBS) at 4°C overnight in a humidified container. Biotinylated goat anti-rabbit immunoglobulin G (IgG) (1:400; Sigma-Aldrich, St Louis, MO, USA) was incubated with the sections for 1 hour at room temperature and detected using a streptavidin-peroxidase complex. The brown colour indicative of peroxidase activity was developed by incubation with 0.1% 3,3′-diaminobenzidine (Sigma-Aldrich) in PBS with 0.05% H₂O₂ for 5 minutes at room temperature. The appropriate positive and negative controls were included in each run of IHC.