Ix71 system

To retrieve ovarian samples from adult mice (8 weeks old) during each of the four stages of the estrous cycle, we performed the vaginal smear tests to identify the specific estrous cycle stages. Briefly, we placed the tip of a plastic pipette filled with 40 μl of phosphate buffered saline after (PBS; Hyclone, USA) into the mouse vagina, and gently flushed the vagina 3–5 times. Vaginal contents (cells and vaginal fluids) from each mouse were collected into a PCR tube and then smeared onto a glass slide. The evaluation of the vaginal smear images was performed under a microscope IX71 system (Olympus, Japan). Proestrus was characterized by the mostly nucleated and some cornified epithelial cells in the smear; estrus by cornified squamous epithelial cells without visible nuclei; metestrus by the predominance of leucocytes and a few nucleated epithelial and/or cornified squamous epithelial cells; and diestrus was characterized by a predominance of leukocytes [24 (link), 25 ]. The reader is directed to further citations for a full description [24 (link), 25 ]. Vaginal smear tests were performed at 08:00 and 20:00 each day. When the stages were identified, the mice were sacrificed via cervical dislocation and their ovaries were collected.

Patient-derived myoblasts were seeded onto 4-well culture slides and were maintained at 37 °C under humidified atmosphere of 5 % CO₂. After 3 days in culture, cells were fixed, permeabilized, and blocked according to standard immunocytochemical protocol. Primary antibody probing was performed at room temperature for 2 h. Secondary antibody probing was performed with 2.5 μg/mL Alexa Fluor 568 (Molecular Probes) at room temperature for 1 h. Mitochondria were co-stained with 0.25 μg/mL MitoTracker Green (Molecular Probes). Stained cells were observed under a fluorescent microscope (IX71 System; Olympus). Primary antibodies used were as follows: 2.5 μg/mL anti-MT-CO1 (Molecular Probes), 2.5 μg/mL anti-COX4 (Molecular Probes).

Cytochemical COX staining was performed as follows: Briefly, undifferentiated and differentiated SH-SY5Y WT and SH-SY5Y ρ⁰ were stained with COX reaction buffer (pH 5.5; 100 mM sodium acetate, 0.1% MnCl₂, 0.001% H₂O₂, 10 mM diaminobenzidine) at 37 °C for 1 h, followed by subsequent incubation with 1% CuSO₄ at 37 °C for 5 min. Stained samples were observed under a microscope (IX71 System; Olympus).