Ultrastructural Analysis of Muscle Mitochondria

The samples that were fixed in 100% ITO-solution were post-fixed in osmium tetroxide (1%), dehydrated afterwards with ethanol/propylene oxide, and embedded in EPON™ 812 (SERVA GmbH, Heidelberg, Germany). Ultrathin sections (60–80 nm) were cut using a Reichert Ultracut S ultramicrotome (Leica Microsystems, Wetzlar, Germany) and contrasted with 4% uranyl acetate and lead citrate. The ultrastructural integrity of the muscle mitochondria was observed with a TEM Zeiss EM 10 C, Carl Zeiss GmbH, Jena, Germany). Pictures were taken with an Image ASP System (SYSPROG, Minsk, Belarus), as previously described [42 (link)]. Using ImageJ software (Scion Image, National Institutes of Health, Bethesda, MD, USA), ten images per mouse were randomly taken at a magnification of 25,000×. Images were analyzed with regards to alterations in the mitochondrial ultrastructure that were categorized as “normal” or “damaged” as follows: Mitochondria with a >50% loss of the cristae, and/or those with >50% disruption of the outer membrane (OMM) were assigned to “damaged” or otherwise to “normal”. Results per mouse were presented as the ‘damaged’ fraction (%) of the total mitochondria count.