Mouse hearts were retrogradely fixed in 2.5% glutaraldehyde in sodium cacodylate buffer, embedded in 2% agarose, post-fixed in buffered 1% osmium tetroxide and stained in 2% uranyl acetate, dehydrated with an ethanol graded series and embedded in EMbed-812 resin as described previously8 (link). Thin sections were cut on an ultramicrotome and stained with 2% uranyl acetate and lead citrate. Images were acquired on a FEI Tecnai G2 Spirit electron microscope equipped with a LaB6 source and operating at 120 kV. Mitochondrial cross-sectional areas were traced and measured using the Multi-Measure ROI tool in ImageJ software (National Institutes of Health). Mitochondrial density was assessed by counting the number of mitochondria versus sarcomeres in a given field.
Protocol detail
Ultrastructural Analysis of Mouse Cardiac Mitochondria
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Agarose
Cacodylate
Citrate
Dehydrated ethanol
Glutaraldehyde
Hearts
Microscope electron
Mitochondria
Mitochondrial
Mouse
Osmium tetroxide
Resin
Sarcomeres
Sodium
Thin sections
Ultramicrotome
Uranyl acetate
Corresponding Organization :
Other organizations : The University of Texas Southwestern Medical Center, Johns Hopkins Medicine, Johns Hopkins University, Advanced Center for Chronic Diseases, University of Chile, Buck Institute for Research on Aging
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Variable analysis
independent variables
- Glutaraldehyde concentration (2.5%)
- Osmium tetroxide concentration (1%)
- Uranyl acetate concentration (2%)
- Mitochondrial cross-sectional area
- Mitochondrial density (number of mitochondria versus sarcomeres)
- Sodium cacodylate buffer
- Ethanol graded series for dehydration
- EMbed-812 resin for embedding
- Staining with uranyl acetate and lead citrate
- Positive control: None mentioned
- Negative control: None mentioned
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