Apoptosis detection system kit

Cells grown on 8-well chamber slides were exposed to OGD. After fixing with 4% (v/v) paraformaldehyde for 15 min, cells were washed with PBS containing 1% (w/v) bovine serum albumin, permeabilized with 0.1% (v/v) Triton-X100, washed with PBS, and incubated for 1 h at 37°C in the dark with an apoptosis detection solution (Apoptosis Detection System kit; Roche Molecular Biochemicals, Mannheim, Germany). In situ-labeled nuclei were observed and photographed using a Nikon C1-Plus laser-scanning TE2000-E confocal microscope.

Cells were seeded onto pNFS and incubated in a humidified 5% CO₂/95% air incubator at 37 °C. After 3 days, pNFSs were stacked in four layers and incubated for 1 day, after which cells were treated with 3 μM DOX or exposed to ionizing radiation (4 Gy). After 1 day, cells were fixed with 3.7% PFA for 15 min, washed with PBS containing 3% (w/v) BSA, and permeabilized with 0.1% (v/v) Triton X-100. After washing with PBS, cells were incubated for 1 h at 37 °C in the dark with an apoptosis-detection solution (Apoptosis Detection System Kit; Roche Molecular Biochemicals, Mannheim, Germany). In situ-labeled nuclei were observed and imaged using a C1-Plus laser-scanning TE2000E confocal microscope (Nikon, Tokyo, Japan).