Glutaraldehyde

As described above, mice were deeply anesthetized and transcardially perfused with PBS; half of the brain was dissected and immersion‐fixed in 4% PFA and stored at 4°C. Later, the brains were postfixed with 4% formaldehyde (Serva, Heidelberg, Germany), 2.5% glutaraldehyde (Science Services, Munich, Germany), and 0.5% NaCl in phosphate buffer pH 7.4 17. Finally, the corpus callosum was postfixed with 2% OsO₄ (Science Services) in 0.1 M phosphate buffer pH 7.3 and embedded in EPON (Serva) after dehydration with ethanol and propylene oxide. EPON blocks with embedded tissue were then trimmed, using a Leica EM TRIM (Leica, Vienna, Austria), to the size of the corpus callosum. In the following, ultrathin sections were stained with an aqueous solution of 4% uranyl acetate followed by lead citrate 18. The pictures were taken in an unbiased random fashion with a Zeiss EM900 electron microscope (Zeiss, Oberkochen, Germany) using a side‐mounted 2k CCD camera (TRS, Waakirchen, Germany). Counting of myelinated and demyelinated axons was performed using NIH ImageJ software (National Institutes of Health, Bethesda, MD, USA); a total area of 10*784 μm² = 7840 μm² was analyzed per mouse and every axon counted. The g ratio was calculated as the ratio between the number of demyelinated axons and the number of myelinated axons. Counting was performed on groups of n = 5 mice.

Animal livers and isolated mitochondria were fixed with 2.5% glutaraldehyde (Science Services GmbH), postfixed with 1% osmium tetroxide, dehydrated with ethanol and propylene oxide, and were embedded in epoxy resin. Sixty-nanometer sections were cut using the Leica EM UC7 microtome (Leica Biosystems) or the Reichert-Jung Ultracut E microtome (now Leica Biosystems). Ultrathin sections were negative-stained with uranyl acetate (Uranyless) and lead citrate. Images were acquired using either a FEI Tecnai-12 electron microscope equipped with a VELETTA CCD digital camera (FEI, Eindhoven, The Netherlands) or using a Jeol 1200 EXII electron microscope (Akishima, Tokyo, Japan) equipped with a KeenViewII digital camera (Olympus, Hamburg, Germany) and processed with the iTEM software package (anlySISFive; Olympus).
For structural analyses, mitochondria were grouped in normally structured mitochondria of the “condensed type”⁵⁹ (link) or in altered mitochondria with marked membrane detachments, matrix condensations, and ballooned cristae. A total of 350–750 mitochondria were included per group of animals.

EHTs were fixed in a mixture of 4% paraformaldehyde and 1% glutaraldehyde (Science Services, Germany) in 0.1 M phosphate buffer overnight at 4 °C. Samples were rinsed three times in 0.1 M sodium cacodylate buffer (pH 7.2–7.4) and osmicated using 1% osmium tetroxide in cacodylate buffer. Following osmication, the samples were dehydrated using ascending ethanol concentrations, followed by two rinses in propylene oxide. Infiltration of the embedding medium was performed by immersion in a 1:1 mixture of propylene oxide and Epon (Science Services, Germany), followed by neat Epon and hardening at 60 °C for 48 h. For light microscopy, semi-thin sections (0.5 μm) with longitudinal orientation were mounted on glass slides and stained for 1 min with 1% toluidine blue. For electron microscopy, ultra-thin sections (60 nm) were cut and mounted on copper grids and stained using uranyl acetate and lead citrate. Sections were examined and photographed using an EM902 (Zeiss) electron microscope equipped with a TRS 2K digital camera (A. Tröndle, Moorenweis, Germany).