Tunel in situ cell death detection kit

Tumor sections were deparaffinized and hydrated. They were incubated with Proteinase K for 15 min and washed in PBS (4 times). The sections were analyzed using a TUNEL In situ Cell Death Detection Kit following the manufacturers' instructions (Sigma-Aldrich) and stained with DAPI. TUNEL-positive cells were quantified in 4 non-overlapping random fields by optical microscopy.

Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was performed using the TUNEL in situ cell death detection kit (Sigma, Missouri, MO) according to the manufacturer’s instructions. Cells were fixed with 10% formalin and exposed to 0.01% Triton X-100 for 2 min on ice. After that, cells were incubated with TUNEL reaction buffer. The nuclei of the cells were counterstained with PI (10 μg/ml) and examined under a fluorescence microscope. For tissue TUNEL staining, the slides were rehydrated, incubated with H₂O₂, and blocked with 10% bovine serum albumin (BSA, Sigma, Missouri, MO, U.S.A.). After blocking, the slides were incubated with TUNEL reaction buffer and PI (1 μg/ml) for 30 min. The number of TUNEL-positive cells was quantified upon observation of ten random fields under 200× magnification and analyzed via Photoshop (Adobe.Photoshop.CS3.Extended v10.0).

After various treatments, HBMECs were planted into 24-well plates. Then, TUNEL In Situ Cell Death Detection Kit (Sigma-Aldrich) was implemented to measure cell apoptosis intensity. HBMECs were coped with TUNEL (5 µM; Sigma-Aldrich) and the cell nucleus was exposed to DAPI and observed by a fluorescent microscope (200× magnification; Leica, Shanghai, China). The experiments were performed with three biological replicates and three technical replicates.

To assess cell apoptosis, hippocampal neurons were planted into 6-well plates. Afterwards, a TUNEL in Situ Cell Death Detection Kit (11684817910; Sigma-Aldrich) was used to assess cell apoptosis. Hippocampal neurons were incubated with TUNEL (5 µM; Sigma-Aldrich) and these cell nuclei were stained with 4′,6-diamidino-2-phenylin-dole (D9542; Sigma-Aldrich). In the last, a fluorescent microscope (Leica, Shanghai, China) was utilised to observe these cells. For hippocampal tissues of rats, five tissue slices of each specimen were taken for apoptosis detection. For each slice, a single region of interest within hippocampal CA1 region was randomly selected to take photos. All counts in the Tunel assay were done by an independent investigator in a blinded manner.