TUNEL assay

To assess the level of apoptotic cell death, TUNEL assay was used, which labels the fragmented DNA. Brains were embedded in OCT compound, then 10-μm sagittal frozen sections were prepared. Sections were post-fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) solution (pH = 7.4) for 20 min. Following a 30-min PBS rinse, sections were blocked in 3% H₂O₂ in methanol for 10 min, then they were permeabilized with a solution of 0.1% Triton X-100 and incubated in 0.1% sodium citrate for 2 min. After washing with PBS, 50 μl of TUNEL reaction mixture was added to each sample (In situ Cell Death Detection Kit, POD, Roche Applied Science, IN, USA) and the sections were incubated for 1 h at 37 °C in a humidified atmosphere. Under the same circumstances, the sections were incubated with Converter-POD solution for 30 min, then washed in PBS, followed by a 10-min signal conversion with a peroxidase reagent. The red apoptotic cells were detected under a light microscope. For quantitative comparison, the TUNEL-positive cells were counted in fifteen fields of view of each brain section (n = 6 mice per group).