Computational protocol: Trans-mitochondrial coordination of cristae at regulated membrane junctions

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Protocol publication

[…] Samples were immediately fixed by immersion in a 2% glutaraldehyde solution in 0.1 M cacodylate, buffer (pH 7.4). After subsequent buffer washes, samples were post-fixed in 2.0% osmium tetroxide for 1 h at room temperature and rinsed in distilled H2O before the in-bloc staining with 2% uranyl acetate. After dehydration through a graded ethanol series, each sample was infiltrated and embedded in EMbed-812 (Electron Microscopy Sciences, Fort Washington, PA). To verify orientation and section quality, 1-μm-thick sections were cut and stained with 1% toluidine blue. Thin sections (90 μm) were mounted on filmed copper grids and stained with uranyl acetate and lead citrate and examined on a JEOL 1010 electron microscope fitted with a Hamamatsu digital camera and AMT Advantage image capture software.We found that glutaraldehyde-only fixation is primordial for preservation of electron density in IMJs, whereas paraformaldehyde is not optimal. Post-staining of ultrathin sections (with uranyl acetate and lead citrate) was not necessary to visualize electron-dense IMJs, which were visible from en-block-only osmium tetroxide-stained specimens. In addition to published images from laboratories worldwide where IMJs and cristae features can be observed (see and associated images), our observations of IMJs in mouse heart and skeletal muscle were reproducible in samples collected in seven different laboratories across eight strains of mice, with tissues processed and imaged in five different imaging facilities/laboratories.For tomography, ~150–300-μm-thick sections were imaged by dual axis image tilt series from −60° to +60° in 1.5 degree increments on a FEI Tecnai 12 microscope equipped with a Gatan US-1000 camera at × 6,500–12,000 magnification using SerialEM. Tomographic volumes were reconsructured within the etomo software package. Analysis and video synthesis were performed using 3Dmod (IMOD 4.7, Boulder Laboratory for 3-D Electron Microscopy of Cells) and Image J (NIH, version 1.47v). […]

Pipeline specifications

Software tools SerialEM, IMOD
Application cryo-EM
Organisms Homo sapiens