Tcs sp5 x confocal microscope

For time-lapse recording, zebrafish embryos were anesthetized in 0.02% tricaine (Sigma), and then embedded in 1.5% low-melting point agarose. Time-lapse images were taken at 30-minute intervals over 15 hours with z-stack collection using a Leica TCS SP5 X confocal microscope; the images were then processed using ImageJ software. High resolution confocal images of the PLL primordium were captured using a Zeiss LSM510 laser scanning confocal microscope (Carl Zeiss, Jena, Germany).

For detection of mitochondrial ion O₂⁻ production, H9c2 myoblasts were stimulated at 37°C with 200 μmol/l palmitate-BSA in the absence or presence of MCP (0.01%). Thereafter, cells were washed and loaded with 5 μmol/l MitoSOX^TM Red for 30 min, at 37°C. The fluorescent signal was analyzed by recording FL2 fluorescence in a Gallius^TM flow cytometer (Beckman Coulter).
To evaluate the mitochondrial transmembrane potential (ΔΨm), H9c2 myoblasts were incubated with 4 μmol/l of Rhodamine 123 for 15 min at 37°C. Stained cells were washed with serum-free medium and stimulated with the indicated doses with palmitate-BSA for 24 h at 37°C. After treatment, cells were washed with PBS and changes in fluorescence were monitored using flow cytometry analysis. In some experiments, before incubation with 200 µmol/l of palmitate-BSA, H9c2 cells were pretreated for 30 min with 3 µmol/l of Triacsin C, an inhibitor of long fatty acid acyl-CoA synthetase (Sigma, St Louis, MO) or with the indicated dose of MCP. Experiments were repeated at least three times. Cells were also visualized on a Leica TCS SP5 X confocal microscope with a ×40 objective.

Immunohistochemistry was performed as described in Pollen et al. [32 (link)]. Briefly, tissue samples were fixed in 4 % paraformaldehyde, cryoprotected in 30 % sucrose, and embedded in a 1:1 mixture of 30 % sucrose and optimal cutting temperature (Thermo Scientific). Cryosections of 20 μm were collected using a Leica CM3050S cryostat. Primary antibody: ADRA2A (1:100, Thermo Scientific, PA1-048). Heat-induced antigen retrieval was performed in 10 mM sodium citrate buffer, pH 6. Binding was revealed using Alexa Fluor™ 488 fluorophore-conjugated secondary antibody (Life Technologies). Images were collected with a Leica TCS SP5 X Confocal microscope.