Bitan lab wiki:TUNEL staining

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TUNEL staining Terminal deoxyribonucleotidyl transferase nick end labeling is mainly detect the DNA fragmentation. This test is mainly used in research purpose for detection of apoptotic or necrotic cell death.

Reagents and accessories:

APO-BrdU-apoptosis detection kit (Invitrogen), cover slips (Fisher Scientific), Poly D-lysine, paraformaldehyde (Sigma),

Protocol:

Primary cortical neurons were grown on poly D-lysine-coated cover slips and treated with peptides for 48 h.

The cells were then washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde.

Cover slips then were treated with ethanol for 30 min at –20ºC, followed by 3×5 min washes with the “wash buffer” supplied in the kit, and incubated with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) DNA labeling solution for 1 h at 37ºC.

The cells were washed and treated with anti-BrdU antibody for 30 min at room temperature in the dark.

Cover slips then were washed with the “rinse buffer” supplied in the kit and counter stained with 1% propidium iodide for 15 min in the dark, washed with deionized water, dehydrated, cleared and mounted on glass slides using glycerol.

Fluorescent signals were visualized using a fluorescence microscope at λex = 480 nm and λem = 530 nm.

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