Ultrastructural Analysis of Mitochondria

Differentiated myotubes of the patient and the control were removed from the culture flask by a scraper and transferred to a conical tube. The medium was removed and cells were fixed with 2% glutaraldehyde solution in PBS at room temperature for 30 min. The samples were post-fixed in 1% osmium tetroxide at room temperature in dark for 1 h, embedded in 2% agar, dehydrated in a series of ethanol, cleared in propylene oxide and embedded in epon plastic blocks (Agar Scientific, UK)^{58 (link)}. The ultra-thin sections were stained with uranyl acetate and lead citrate, analyzed with a TEM and attached digital camera (TEM, JEM1400, Japan, GATAN, Germany). The length and width of mitochondria (at least 50 mitochondria per section) were measured with DigitalMicrograph (Gatan Inc., USA) software^{6 (link),59 (link),60 (link)}.

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Aksu-Menges E., Eylem C.C., Nemutlu E., Gizer M., Korkusuz P., Topaloglu H., Talim B, & Balci-Hayta B. (2021). Reduced mitochondrial fission and impaired energy metabolism in human primary skeletal muscle cells of Megaconial Congenital Muscular Dystrophy. Scientific Reports, 11, 18161.

Publication 2021

JEM1400 DigitalMicrograph

Agar Camera Cells Citrate Dehydrated ethanol Epon Glutaraldehyde Mitochondria Myotubes Osmium tetroxide Patient Propylene oxide Thin sections Uranyl acetate

Corresponding Organization :

Other organizations : Hacettepe University

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Variable analysis

independent variables

Differentiated myotubes of the patient
Differentiated myotubes of the control

dependent variables

Length of mitochondria
Width of mitochondria

control variables

Fixation with 2% glutaraldehyde solution in PBS at room temperature for 30 min
Post-fixation in 1% osmium tetroxide at room temperature in dark for 1 h
Embedding in 2% agar
Dehydration in a series of ethanol
Clearing in propylene oxide
Embedding in epon plastic blocks
Staining with uranyl acetate and lead citrate
Analysis with a TEM and attached digital camera (TEM, JEM1400, Japan, GATAN, Germany)
Measurement of mitochondrial length and width with DigitalMicrograph (Gatan Inc., USA) software

controls

Differentiated myotubes of the control

Annotations

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