Coolsnaphq2

Oocyte in vitro maturation assays were performed as previously described (Brunet et al., 2008 (link)). Briefly, oocytes were collected from ovaries of 10- or 26-week-old hmmr^+/+ and hmmr^m/m mice and placed in M2 medium pre-warmed to 37°C and supplemented with 4 mg/ml BSA and 1 mM milrinone. For video microscopy of oocyte meiotic maturation, oocytes were transferred to a Ludin Chamber containing M2 medium with 4 mg/ml BSA. Time-lapse images were acquired using a Photometrics CCD camera (CoolSnap HQ2) mounted on a Leica HC PL APO 20×/0.7 NA objective enclosed in a thermostatic chamber (Life Imaging Service). Images were taken every 15 min for 18–20 h at 20× magnification. Metamorph 7.0 (Universal Imaging) and ImageJ (NIH) software were used for image analysis.

Senescence-associated beta-galactosidase (β-Gal) staining was performed according to the manufacturer's protocols (Cell Signaling Technology). At the end of treatments, cells were fixed for 15 minutes in 1x fixative solution (formaldehyde-glutaraldehyde mix), followed by PBS washes, and incubated overnight with β-Gal staining solution in a dry incubator at 37°C. Cells were viewed under brightfield microscopy (Leica BM5500B Microscope; Leica Biosystems, Wetzlar, Germany) for blue color development and were photographed using a Photometrics CoolSNAP HQ² camera and associated Leica Application Suite software v4.1.

Fixed cells were obtained after incubation with 3.2% paraformaldehyde (PFA) for 10 min at room temperature, washed with PBS, incubated with PBS-0.1 M NH₄Cl for 5 min and then washed with PBS. Finally, cells were permeabilized in 0.1% Triton X-100 for 3 min and blocked with 1% BSA during 10 min. Fixed cells were mounted using mowiol mounting agent and visualized using a Leica DMRA and a CoolSnapHQ2 camera, 100× objective NA 1.25 oil Ph 3 CS (HCX PL APO) and analyzed with the Metamorph software. 3D stacks were acquired and deconvolved to build a projection on one plane using Image J.
For myotube imaging, images were acquired on a Zeiss LSM880 Airyscan confocal microscope (MRI facility, Montpellier). Excitations sources used were: 405 nm diode laser, an Argon laser for 488 nm and 514 nm and a Helium/Neon laser for 633 nm. Acquisitions were performed on a 63 ×ȉ/1.4 objective. Multidimensional acquisitions were performed via an Airyscan detector (32-channel GaAsP photomultiplier tube (PMT) array detector). Images are presented as a z-projection of all planes.