TUNEL Staining for Apoptosis Detection

TUNEL staining was performed using an In Situ Cell Death Detection Kit (Roche, Mannheim, Germany), according to previously described methods [15 (link),17 (link)]. The sections were postfixed in ethanol-acetic acid (2:1), and then were rinsed. The sections were then incubated with proteinase K (100 μg/mL), rinsed, incubated in 3% H₂O₂, and permeabilized with 0.5% Triton X-100. After the sections were rinsed again, the sections were incubated in the TUNEL reaction mixture. After the sections were rinsed, the sections were visualized using Converter-POD with 0.03% DAB. Mayer hematoxylin (DAKO, Glostrup, Denmark) was used as a counterstain. The sections were mounted onto gelatin-coated slides, and the coverslips were mounted using Permount (Fisher Scientific).