Lsm 780 metaconfocal laser scanning microscope

Images were captured with a 20× objective using an LSM 780 Metaconfocal laser scanning microscope (Carl Zeiss MicroImaging, Inc., Jena, Germany). For OLIG2 and CNP cell counts, 10–15 fields were taken per condition. For MBP area quantification, four fields were taken per mouse. Three to four mice were included per treatment condition. MBP+ area and OLIG2+/CNP+ cell counts were quantified using ImageJ (Liu et al., 2016 (link); Rusielewicz et al., 2014 (link)). An unpaired Student’s t test or one-way ANOVA was performed to assess statistical differences between conditions as indicated in figure legends.

Immunofluorescence was performed as described (Zhang et al., 2015 (link)). For immunofluorescent staining, oocytes were fixed in 4% paraformaldehyde in PBS for 30 min at room temperature. After permeabilization with 0.5% Triton X-100 for 20 min, they were blocked in 1% BSA in PBS for 1 h at room temperature. For staining of CenpH, ACA and γ-tubulin, oocytes were incubated overnight at 4°C with anti-CenpH (1:100), anti-ACA (1:40) or anti-γ-tubulin (1:200) antibodies. After three washes in washing buffer, oocytes were incubated with FITC-conjugated goat-anti-mouse IgG (1:100) or cy5-conjugated goat anti-human IgG (1:500) or TRITC-conjugated goat anti-mouse IgG (1:100) for 2 h at room temperature. For α-tubulin staining, oocytes were incubated with anti-α-tubulin-FITC antibodies for 2 h at room temperature, oocytes were then washed three times in wash buffer and co-stained with Hoechst 33342 (10 mg/ml in PBS) for 15 min. These oocytes were mounted on glass slides and examined with an LSM 780 META confocal laser-scanning microscope (Zeiss).

Early apoptosis and late apoptosis/necrosis were detected using an Annexin-V FITC/PI Apoptosis Detection kit according to the manufacturer’s instruction. Briefly, cardiomyocytes were seeded into a 12-plate at a density of 5×105/well, after the indicated treatment, adherent cells were enzymatically digested for 50S with 0.25% trypsin and collected together with floating dead cells. Approximately 1×106 cells were washed twice with cold PBS, and then resuspended in 200μl cold 1×binding buffer containing 5μl of Annexin V-FITC and 5μl of PI. Cells were incubated at room temperature for 15 min in the dark and fluorescence was analyzed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA). At least 10,000 events were recorded. Apoptotic cells were expressed as a percentage of the total number of cells. Apoptosis of cardiomyocytes were analyzed with an in situ cell death detection kit as described in our previous publication [12 (link)]. The staining was observed under a Zeiss confocal laser scanning microscope (Carl Zeiss, LSM 780 Meta Confocal Laser Scanning Microscope). The experiment was repeated three times.