TUNEL assay for apoptosis of neurons

After dewaxing, the cells were sliced into 5 sections; each section was diluted with 1% protease K. Next, the sections were added with 0.3% H₂O₂ methanol solution, dripped with TUNEL reaction solution and incubated in a 37 °C humidity chamber for 1 h. When the cells exhibited brown-yellow nuclei under microscope investigation, the reaction was terminated with distilled water. Afterwards, the sections were counterstained with hematoxylin, and observed under an optical microscope (× 400), with 10 fields of vision selected from each section. The positive cells and podocytes were counted respectively. The cells displaying brown-yellowish nuclei were regarded as positive apoptotic cells while the cells with blue nuclei were considered to be normal cells. The average number of positive apoptotic cells as well as the normal cells was subsequently calculated. The apoptotic index (AI) was reflected by the ratio of the number of positive apoptotic cells to that of the normal cells.