Peroxidase blocking system

The mice were killed by carbon dioxide (CO₂) inhalation, and the tumors were removed and frozen in Tissue-Tek O.C.T. compound at −80°C. A standard streptavidin horseradish immunoperoxidase method was used for human CD3 staining as described previously [16 (link)]. Briefly, 7-μm cryosections were fixed in cold acetone for 5 minutes at 4°C and blocked with a peroxidase blocking system for 10 minutes (Dako, Carpinteria, CA, USA). Routine immunostaining was carried out with an initial 30-minute incubation with 10% normal goat serum at room temperature (RT), primary rabbit anti-human CD3 for T cells (RM-9107; Thermo Scientific, Waltham, MA, USA) at 1:100 dilution at RT for 45 minutes, secondary biotinylated goat anti-rabbit antibody at 1:200 dilution at RT for 30 minutes and streptavidin-biotinylated horseradish peroxidase complex reagent (Dako) at RT for 30 minutes, followed by washing three times for 5 minutes each after each incubation. Sections were then exposed to DAB + chromogen (Dako) for 5 minutes at RT, counterstained with hematoxylin, dehydrated, cleared and mounted.