Lsm700 meta confocal laser microscope

The HCS Mitochondrial Health Kit (H10295, Thermo Fisher Scientific) was used to measure mitotoxicity and cytotoxicity simultaneously in the same cell. Briefly, cell staining solution was prepared by adding 10.5 μl of the MitoHealth stain solution and 2.1 μl of Image-iT® DEAD Green™ viability stain to 6 ml complete medium according to the manufacturer’s protocol. The neuronal cultures were incubated with the cell staining solution for 30 min under culture conditions, followed by adding the counterstain/fixation solution for 15 min at room temperature. The red MitoHealth stain corresponding to mitochondrial membrane potential (MMP) and DEAD Green (cytotoxicity) were captured under a LSM700 Meta confocal laser microscope (Carl Zeiss).

As previously reported by our laboratory (Zhang et al., 2008 (link)), floating coronal brain sections were washed with PBS and then blocked with 10% normal donkey serum at RT for 1 h. After blocking, the sections were incubated overnight at 4°C with following primary antibodies: NeuN (1:200), Microtubule associated protein 2 (MAP2, 1:100), Ionized calcium-binding adapter 1 (Iba-1, 1:200), NQO1 (1:100), SOD2 (1:100), HO-1 (1:100), Glial fibrillary acidic protein (GFAP, 1:200, Proteintech), Cleaved Caspase-3/9 (1:100, Cell Signaling) and Malondialdehyde (MDA, 1:100, Novus Biologicals). After washes, sections were incubated with corresponding secondary antibodies and mounted with DAPI. Images were captured by a LSM700 Meta confocal laser microscope (Carl Zeiss).