Click it plus tunel kit

The terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay was used to identify apoptotic cells with nuclear DNA fragmentation in brain ischemic core areas. Staining was performed according to the manufacturer’s instructions (Click-iT Plus TUNEL Kit, Thermo Fisher, Waltham, MA). Briefly, brain sections adjacent to those used for immunohistochemistry were incubated with proteinase K (15 min, RT) and then rinsed with PBS. After incubation with TdT reaction buffer (10 min) and TdT reaction mixture (1 h at 37 °C), sections were washed and incubated with Click-iT Plus reaction cocktail containing Alexa Fluor 488 (30 min at 37 °C). Finally, sections were counterstained with DAPI.

CMVECs were previously seeded on poly-l-lysine-treated coverslips. Fixed cells were stained using a terminal deoxynucleotidyl transferase-mediated dUTPbiotin nick end labeling (TUNEL) kit according to the manufacturer's instructions (Click-iT Plus TUNEL Kit, Thermo Fisher), and cell nuclei were stained with DAPI, as previously reported [18 (link)]. For quantification, the TUNEL-positive cells were counted in at least five randomly chosen visual fields in three independent samples under a fluorescence microscope (Olympus). The total number of cells was counted using DAPI staining, and the average ratio of TUNEL-positive cells was calculated.