Anti cd8

For histologic examination, mice were intra-dermally challenged five days after the last sensitization. After 20 min, 24 h, and 48 h, the mice were sacrificed at the indicated time and the abdominal skins from the challenge sites were removed and placed in 10% formalin overnight at room temperature. The removed skin samples were 2 mm larger than the lesions, and sized varied from 4–7 mm in diameters (16–49 mm²). Briefly, the tissues were embedded in paraffin, cut into 5-μm sections, de-paraffinized, dehydrated, and stained with H and E. Moreover, sections were further stained with rabbit anti-CD4 (1∶800 dilution) or anti-CD8 (1∶400 dilution) polyclonal antibodies (Bioss, MA, USA). To detect CD marker positive cells, the sections were incubated with peroxidase-conjugated goat anti-rabbit IgG and stained in a substrate solution containing DAB in Bond automatic system (Leica, Newcastle, UK). Inflammatory cell infiltrates were examined by light microscopy and corresponding images were shot by the Olympus BX51 microscopic/DP71 Digital Camera System (Nagano, Japan).