Vectashield mounting medium containing dapi

Single myofibres were isolated as previously described^{34 (link),44 (link)}. Muscles were fixed with 4% paraformaldehyde for 2 days. Fixed muscle was washed in phosphate-buffered saline (PBS pH 7.2) and fibre bundles mechanically separated in 40% NaOH. Muscle bundles were macerated in 40% NaOH solution for 3 h at room temperature and then shaken for 10 minutes to individualize single myofibres. Alkali maceration led to isolate muscle fibres free of attached cells. Isolated myofibres were rinsed twice with PBS for neutralization. For myonuclei imaging, isolated myofibres were then mounted on glass slides in Vectashield mounting medium containing DAPI (Vector Laboratories, UK). Isolated fibres were analyzed by confocal laser scanning microscopy using an upright FV-1000 confocal laser scanning microscope (Olympus, France). Z-series from the top to the bottom of fibres were sequentially collected with a step of 0.5 µm between each frame. For immunostaining, muscles were fixed with 0.5% paraformaldehyde for 3 h, washed in PBS pH 7.2 and mechanically dissociated.

For hematoxylin and eosin staining, testes, epididymis and ovaries were fixed in 10% formalin or Bouin solution and embedded in paraffin. Sections were prepared on CREST-coated slides (Matsunami) at 6 μm thickness. The slides were dehydrated and stained with hematoxylin and eosin.
For Immunofluorescence staining, testes were embedded in Tissue-Tek O.C.T. compound (Sakura Finetek) and frozen. Cryosections were prepared on the CREST-coated slides (Matsunami) at 8 μm thickness and then air-dried and fixed in 4% paraformaldehyde in PBS at pH 7.4. The serial sections of frozen testes were fixed in 4% PFA for 5 min at room temperature and permeabilized in 0.1% TritonX100 in PBS for 10 min. The sections were blocked in 3% BSA/PBS or Blocking One (Nakarai), and incubated at room temperature with the primary antibodies in a blocking solution. After three washes in PBS, the sections were incubated for 1 h at room temperature with Alexa-dye-conjugated secondary antibodies in a blocking solution. PNA lectin staining was performed using Lectin from Arachis hypogaea, FITC conjugate (1:1000, Sigma-Aldrich L7381). TUNEL assay was performed using MEBSTAIN Apoptosis TUNEL Kit Direct (MBL 8445). DNA was counterstained with Vectashield mounting medium containing DAPI (Vector Laboratory). Statistical analyzes, and production of graphs and plots were done using GraphPad Prism9 or Microsoft Excel.

Ca Ski cells were seeded on slides in 60 mm dishes and transfected with the indicated siRNA. After 72 h, cells were fixed with 3.7% paraformaldehyde in PBS for 15 min and permeabilized with 0.04% NP-40/PBS. Cells were incubated with 0.1M Glycine and 5% BSA solution for 1h at room temperature for blocking purposes. Then, cells were incubated overnight at 4 °C with anti-p53 (Santa Cruz Biotechnology) and anti-SNAT1 (Cell signaling) antibodies. After several washes with PBS 1×, cells were incubated with Alexa fluor 488 donkey anti-mouse (Invitrogen, Waltham, MA, USA) and Alexa fluor Rhodamine Red-X goat anti-rabbit (Invitrogen, Waltham, MA, USA), respectively, for 2 h at 4 °C. Slides were washed and mounted with Vectashield mounting medium containing DAPI (Vector Laboratories, Newark, CA, USA). Slides were visualized with a confocal microscope (Zeiss LSM 710 DUO, Oberkochen, Baden-Wurttemberg, Germany) with lasers giving excitation lines at 488 and 594 nm. Around twenty fields were observed for each treatment and representative images were acquired. Data of three independent experiments were collected with a 63X objective oil immersion lens. In addition, the same protocol was carried out to evaluate SNAT1 in C-33 A stably transfected cells.