Fluorchem 5500 imaging station

Total lipids from muscle homogenates (1.25 mg) were extracted as previously described^{43 (link)} and the lipid extract was spotted onto high-performance thin layer chromatography plates (HPTLC; 5633-5, EMD Chemicals, Darmstadt, Germany) using a chloroform:methanol:acetic acid:water (100:75:7:4) solvent system to separate individual phospholipids^{4 (link)}. A standard curve (0.2, 0.4, 0.8, 1.6 μg) of bovine heart cardiolipin (C0563, Sigma Aldrich, MO, USA) was also loaded onto each HPTLC plate, and after allowing the solvent to run up each plate for 45 min, the plates were then charred at 180 °C with a 10% (w/v) copper sulfate in 8% phosphoric acid solution for 15 min^{44 (link)}. Images of the HPTLC plates were captured using a CCD camera on a Fluorchem 5500 imaging station (Alpha Innotech, CA, USA) under reflective white light. Densitometry analyses were then performed using ImageJ (National Institutes of Health, MA, USA) and the standard curve of bovine heart cardiolipin was used to calculate the absolute CL concentration (per 1.25 mg of muscle) in plantaris and soleus.

Total lipids from plantaris homogenates (1.25 mg) were extracted as previously described [18 (link)] and were subsequently spotted onto high-performance thin layer chromatography plates (HPTLC; 5633–5, EMD Chemicals, Darmstadt, Germany) and individual phospholipids were separated using a chloroform:methanol:acetic acid:water (100:75:7:4) solvent system [4 (link)]. A standard curve (0.5, 1.0, 2.0, 4.0 μg) of purified PC (P3556, Sigma Aldrich, MO, USA), and a standard curve (0.2, 0.4, 0.8, 1.6 μg) of purified PE (P7943, Sigma Aldrich) were also loaded onto each HPTLC plate. After allowing the solvent to run up each plate for 45 min, the plates were then charred at 180 °C with a 10% (w/v) copper (II) sulfate in 8% phosphoric acid solution for 15 min [19 (link)]. Images of the HPTLC plates were captured using a CCD camera on a Fluorchem 5500 imaging station (Alpha Innotech, CA, USA) under reflective white light. Densitometry analyses were then performed using imageJ (National Institutes of Health, MA, USA) and the standard curve of PC and PE (Fig. 1) were used to calculate the absolute PC and PE concentrations (per 1.25 mg of muscle) in plantaris.Fig. 1

Representative images of PC (a) and PE (b) standard curves that were loaded onto each HPTLC plate