Anti mouse gr 1 antibody

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.

Samples of imiquimod-applied mouse back skin were harvested. They were formalin-fixed and stained with hematoxylin and eosin. For immunohistochemistry, mouse skin was snap-frozen and stored at –80 °C. Cryosections were fixed with cold acetone for 5 min and incubated overnight at 4 °C with anti-mouse major histocompatibility complex class II antibody (1/50 dilution, Abcam, Cambridge, UK), anti-mouse CD3 antibody (1/100 dilution, Abcam), anti-mouse Gr-1 antibody (1/100 dilution, BD Pharmingen, San Diego, CA) or anti-mouse F4/80 antibody (1/100 dilution, Bio-Rad, Hercules, CA). Tissues were subsequently stained with an avidin-biotin peroxidase complex using a Vector ABC staining kit (Vector Laboratories, Burlingame, CA, USA). Diaminobenzidine was used for visualizing the staining, and counterstaining with Mayer hematoxylin was performed according to the manufacturers’ instructions. Stained cells were counted in 10 random grids under high original magnification (×400) power fields of a light microscope. Each section was examined independently by two investigators in a blinded manner.