Cellular apoptosis in brain tissues was measured using One Step TUNEL Apoptosis Kits (Beyotime, China). In brief, brain tissue sections were fixed in 4% paraformaldehyde at room temperature for 10 min, washed with PBS, and further fixed in paraformaldehyde plus acetic acid for 5 min at 4°C. After that, they were washed with PBS, equilibrated in equilibration buffer, and incubated with TUNEL reaction mixture containing terminal deoxynucleotidyl transferase (TdT) at 37°C for 1 h. The negative control was treated similarly and incubated with TUNEL reaction mixture without TdT. The TUNEL-positive cells were examined and photographed at ×400 magnification under an epifluorescence microscopy Nikon Eclipse 80i and presented as the percentage of the total cells.
The apoptosis of SH-SY5Y cells was detected by Hoechst 33258 staining using a commercial kit (Sigma‐Aldrich, St. Louis, MO, USA) following the guideline and flow cytometry. For Hoechst 33258 staining, SH-SY5Y cells were fixed in 4% paraformaldehyde for 15 min. After washed with PBS, cells were incubated with Hoechst 33258 staining solution at room temperature for 5 min and observed under a fluorescence microscope. The apoptotic cells were counted measured in a blinded manner and represented as the proportion to the total cells.
The apoptosis of SH-SY5Y cells was detected by Hoechst 33258 staining using a commercial kit (Sigma‐Aldrich, St. Louis, MO, USA) following the guideline and flow cytometry. For Hoechst 33258 staining, SH-SY5Y cells were fixed in 4% paraformaldehyde for 15 min. After washed with PBS, cells were incubated with Hoechst 33258 staining solution at room temperature for 5 min and observed under a fluorescence microscope. The apoptotic cells were counted measured in a blinded manner and represented as the proportion to the total cells.