TUNEL assay for measuring cellular apoptosis in the mouse brain

The TdT-mediated dUTP-biotin nick end-labeling (TUNEL) assays were performed by using the TUNEL cell apoptosis detection kit via a green FITC-labeled fluorescence detection approach (catalog: KGA7071, KeyGEN BioTECH Company). Mouse cardiac perfusion was performed to make paraffin slides containing mouse hippocampal tissue. After dewaxing the paraffin sections of mouse hippocampal tissue, 100 μL (20 μg/mL) of the proteinase K working solution was added and washed with PBS 3 times at room temperature for 30 min. Then, the slide was incubated in 100 μL DNase I at 37 °C for 30 min and washed with PBS. After the slide was dried, 50 μL TUNEL reaction mixture (50 μL TdT + 450 μL fluorescein-labeled d UTP solution) was added to the specimens, which were incubated at 37 °C for 60 min and then washed with PBS. DAPI was added dropwise to protect the specimens for 10 min, and the specimens were stained and washed with PBS. The excess DAPI was washed off, the liquid on the slide was blotted with absorbent paper, and the image was observed under a fluorescence microscope with a sealing sheet containing an anti-fluorescence quenching agent. After the nuclei were stained with DAPI, blue fluorescence was displayed, and the apoptotic cells were stained with the TUNEL reaction mixture to show green fluorescence.