Dab peroxidase substrate

Embryos were fixed with PFA, permeabilized with pre-chilled acetone for 10 min, washed in phosphate-buffered saline (PBS) with 0.1% Tween-20 (PBSTw), and blocked with 10% normal goat serum (NGS) in PBS with 0.8% Triton X-100 (PBSTx) for 1 h at room temperature. Then, embryos were incubated overnight at 4 °C in the anti-phospho histone H3 polyclonal antibody (Millipore; 1:300 in 1% NGS/PBSTx). After washing four times for 10 min each in PBSTw, the embryos were incubated overnight with the secondary antibodies (anti-rabbit IgG peroxidase conjugated, 1:300 in 1% NGS/PBSTx). Embryos were washed five times and incubated with DAB peroxidase substrate (Roche). The color reaction was stopped by washing with PBSTw, fixed for 30 min in 4% PFA, washed in PBSTw, and cleared in 70% glycerol/PBSTw.

Liver tissue specimens were embedded in paraffin and sectioned at 5 μm for processing by the TUNEL method using a commercial kit, using DAB peroxidase substrate (Roche Molecular Biochemicals, Meylan, France) and counterstained with methyl green. Specimens were evaluated by microscopy at high power magnification ( × 100) in a blinded manner. A total of 30 random fields were counted for each TUNEL-stained tissue sample. TUNEL assays on primary hepatocytes were performed following exactly the same procedure as we previously described.^{43 (link), 44 (link)}