Lead citrate

Muscular biopsy was performed in the proband of family 2 to observe the ultrastructure of mitochondria in muscular tissue. Muscular tissue was fixed in 2.5% glutaraldehyde (Sangon Biotech, China) in PBS at 4°C for 2 h. Then, the tissue block was washed three times with PBS, and the block embedded with agar were fixed again with 1% osmium tetroxide for 2 h then washed three times with PBS. The tissue block was dehydrated with an ethanol series and fixed again with Epon 812 (Sangon Biotech, China). The ultrathin (70 nm) sections were collected on Formvar/carbon‐coated nickel grids (Sangon Biotech, China), and the grids were stained with 2.5% uranyl acetate (Sangon Biotech, China) for 7 min followed by lead citrate (Sangon Biotech, China) for 2.5 min, and then the sections were observed with a JEM‐1011 electron microscope (JEOL, Japan).

The hippocampal tissue sections at 1 mm³ were fixed in 40 g/l of glutaraldehyde for 1 h and washed 3 times with 0.1 mol/l of phosphate buffer (pH 7.4) for 5 min each time. The tissue sections were fixed with 1% osmium acid for 2-3 h and were washed 3 times with 0.1 mol/l of phosphate buffer (pH 7.4) for 5 min each time. The tissue sections were dehydrated by gradient ethanol, immersed in a mixture of acetone and an equal amount of Epon812 for 3 h, embedded in Epon812, and polymerized at 60°C for 48 h. Thereafter, the sections were stained with 3% uranium acetate and lead citrate (all from Sangon Biotech) at room temperature for 15 min, respectively, and were observed and photographed under a TEM (JEM-1200 EX; JEOL, Ltd.).