Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay

Detection of individual apoptotic cells in formalin-fixed liver sections was performed using In Situ Cell Death Detection Kit, Fluorescein (Roche). Briefly, paraffin-embedded liver sections were deparaffinized, rehydrated, and antigen retrieved. The DNA strand breaks labeling was conducted following the manufacturer’s instructions. Sections were counterstained with DAPI (Vector Laboratories). The pictures of TUNEL-positive cells were obtained by using a LEICA DM2000 fluorescence microscope and analyzed using the Olympus cellSens Standard software. At least 10 different areas per sample were counted for TUNEL-positive cells by 2 staff scientists in a blinded fashion.