Streptavidin biotin peroxidase complex techniques

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.

Tumors from mice were fixed in 10% neutral buffered formalin overnight and embedded in paraffin. Paraffin‐embedded blocks were cut into 5‐μm‐thick sections and stained with hematoxylin and eosin (HE). Immunostaining was performed using streptavidin–biotin–peroxidase complex techniques (Nichirei, Tokyo, Japan). Consecutive cryostat tissue sections (6 μm) were mounted on glass slides and fixed in 99.5% ethanol for 20 min. After blocking with normal rabbit serum, the sections were stained with anti‐mouse Gr‐1 antibody (BD Biosciences). The sections were counterstained with methyl green. Positive cells were counted in 10 representative high‐power fields (400×) under a microscope.