Polyclonal Rabbit Anti-Human Myeloperoxidase (Dako Omnis)

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The antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, California, USA). Formalin-fixed, paraffin-embedded samples were cut into 4-μm sections and each tissue section was deparaffinized and rehydrated with graded ethanol. Antigen retrieval was performed using EDTA antigen repairing buffer (pH 8.0) in microwave for 13 minutes. After natural cooling, tissues were placed in phosphate-buffered saline (PBS) at pH 7.4 twice for 5 minutes each. Tissues were then incubated with 3% hydrogen peroxide solution at room temperature for 25 minutes in the dark. Subsequently, slides were incubated with an antibody against MPO (Dako, Denmark; 1:500) overnight at 4 °C in a humid chamber. Then tissues were incubated in an anti-goat secondary antibody (Dako, Denmark; 1:1000) for 30 minutes at 25 °C. Finally, sections were counterstained with hematoxylin. Under a light microscope, the positive cells were defined as those with dark brown nuclei. The numbers of MPO positive cells were counted in 10 randomly selected fields at 40x magnification. The results were expressed as the average number of positive-stained cells per high-power field (HPF).

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