To assess myelin sheath thickness, cocultures were fixed with 2% glutaraldehyde overnight at 4°C, followed by postfixation with 1% osmium tetroxide for 30 min at room temperature. After the samples had been washed three times with ultrafiltered water, they were dehydrated in an ethanol series (50, 70, 80, 85, 90, 95, and 100%). Using an ultramicrotome (Ultra Cut C; Leica, Wetzlar, Germany), samples were sectioned and mounted on copper grids. Grids were contrast stained with uranyl acetate and lead citrate. Images were captured by cryo-TEM (Tecnai F20 Cryo; FEI, Hillsboro, OR, USA). To quantitatively determine the relative thickness of individual remyelinated nerves, the g-ratio was analyzed with a g-ratio calculator plug-in for ImageJ software (available at https://imagej.nih.gov/ij/ ), which allowed semiautomated analysis of randomly selected nerve fibers (53 (link), 54 (link)). At least seven remyelinated axons per group were randomly selected for measurement, and the mean g-ratio was calculated for each group by averaging across all individual g-ratios.
Protocol detail
Quantifying Remyelinated Nerve Thickness
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Axons
Citrate
Cocultures
Copper
Dehydrated ethanol
Glutaraldehyde
Myelin sheath
Nerve fibers
Nerves
Osmium tetroxide
Ultramicrotome
Uranyl acetate
Corresponding Organization : Seoul National University
Other organizations : Korea Institute of Brain Science, Korea Institute of Science and Technology, Korean Association Of Science and Technology Studies
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Variable analysis
independent variables
- Concentration of glutaraldehyde used for fixation (2%)
- Duration of glutaraldehyde fixation (overnight)
- Temperature of glutaraldehyde fixation (4°C)
- Concentration of osmium tetroxide used for postfixation (1%)
- Duration of osmium tetroxide postfixation (30 min)
- Temperature of osmium tetroxide postfixation (room temperature)
- Number of washes with ultrafiltered water (three times)
- Concentrations of ethanol used for dehydration (50%, 70%, 80%, 85%, 90%, 95%, and 100%)
- Thickness of myelin sheath (quantified using g-ratio)
- Coculture system used for the experiment
- Cryo-TEM (Tecnai F20 Cryo) used for imaging
- ImageJ software with g-ratio calculator plugin used for analysis
- Positive control: Not explicitly mentioned.
- Negative control: Not explicitly mentioned.
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