Rat anti mouse cd4 and cd8

Immunostaining was performed using the streptavidin-biotin-peroxidase complex techniques (Nichirei, Tokyo, Japan). Consecutive cryostat tissue sections (5 μm) were mounted on glass slides and fixed in 99.5% ethanol for 20 min. After blocking with normal rat serum, the sections were stained with rat anti-mouse CD4 and CD8 (BD Biosciences). Negative controls without primary antibodies were examined in all cases. The sections were counterstained with methylgreen.
Immunohistochemical staining of tumor-draining lymph nodes (TDLNs) for Rat IgG (DTA-1) was performed using the streptavidin-biotin-peroxidase complex techniques (Nichirei). Immunohistochemical scoring criteria defined as the following, 3+: Circumferential membrane staining that is complete, intense, and within >10% of lymph node cells. 2+: Circumferential membrane staining that is incomplete and/or weak/moderate and within >10% of lymph node cells, or complete and circumferential membrane staining that is intense, and within ≦10% of lymph node cells. 1+: Incomplete membrane staining that is faint/barely perceptible and within >10% of lymph node cells. 0: No staining is observed, or membrane staining that is incomplete and is faint/barely perceptible and within ≦10% of lymph node cells. The histologic scoring was evaluated by a pathologist (N. H.) in a blinded manner.