Anti mouse cd3 antibody

Tumor tissue samples embedded in Tissue-Tek optimum cutting temperature (OCT) were sectioned by a microtome-cryostat (8 μm). Formalin fixed, paraffin-embedded tissue specimen was prepared for 5 μm sections. Frozen sections were fixed in HistoChoice MB tissue Fixative solution (Ambresco, Cleveland, OH, USA). Subsequently in both frozen and paraffin-embedded sections, peroxidase activity was inhibited by 3% hydrogen peroxide. Additionally, samples were blocked using SuperBlock blocking buffer (Thermo Fisher Scientific). Anti-mouse CD45 antibody, anti-mouse Ly6G/Ly6C antibody, and anti-mouse CD3 antibody (Abcam, Cambridge, MA, USA) were used for immunohistochemistry staining. The signal was developed by diaminobenzidine (DAB) substrate (Dako, Santa Clara, CA, USA).

The samples of imiquimod-applied mouse back skin were harvested. They were formalin-fixed and stained with hematoxylin and eosin. For immunohistochemistry, the mouse skin was embedded in an optimal cutting compound, snap-frozen in liquid nitrogen and stored at −80 °C. Cryosections were fixed with cold acetone for 5 min and incubated overnight at 4 °C with anti-mouse MHC class II antibody (1/50 dilution, Abcam, Cambridge, UK) and anti-mouse CD3 antibody (1/100 dilution, Abcam). Tissues were subsequently stained with an avidin-biotin peroxidase complex using a Vector ABC staining kit (Vector Laboratories, Burlingame, CA). 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. In some experiments, 5 μm-thick tissue sections from freshly frozen samples were stained with fluorescein isothiocyanate (FITC)-conjugated IL-17A (BioLegend, San Diego, CA, USA) and FITC-conjugated IFN-α (Biorbyt, Cambridge, UK), or FITC-conjugated IL-10 (BioLegend).

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.