Ultracut e microtome

Twelve weeks after orthotopic forelimb transplantation, all animals were sacrificed and intracardially perfused with 4% paraformaldehyde. A 5-mm segment of the median nerve was harvested at 5-10 mm distal to the nerve coaptation for histomorphological assessment.
Nerves were fixed in 3% paraformaldehyde + 2% glutaraldehyde in 0.1M Sorensen's Phosphate Buffer fixing SOLUTION. The nerves were then transferred to osmium tetroxide for postfixation, embedded in plastic and sectioned with an Ultracut E microtome (Reichert Technologies, Depew, NJ) to obtain 1-mm-thick cuts. Sections were stained with toluidine blue 1% (in 1% sodium TETRABORATE) to allow for visualization of myelin-sheathed axons. One image of each nerve was taken at 10× magnification, and five representative images of each nerve were taken at 100× magnification using a Nikon (Melville, NY) E500 microscope system. Axon counts and density were then measured using ImageJ SOFTWARE (National Institutes of Health, Bethesda, MD). One hundred fifty axons of each nerve were measured for average G-ratio, myelin thickness, axon diameter and fiber diameter and counted as n = 1.

For 2D EM, ultrathin sections (60-70 nm), sized up to ∼2 × 1.5 mm, were cut with an Ultracut E microtome (Reichert-Jung) or PowerTome (RMC) using an ultra 35°diamond knife for minimal compression of sections (Harris et al., 2006) (Diatome) and stretched using xylene vapor. The grids were picked at their short edge with a fine forceps, dipped into absolute ethanol, then 10 times into distilled water to achieve smoothening of the pioloform film, and then inserted into the water trough of the diamond knife (Supplementary Fig. 3). Sections were placed on the pioloform film by attachment of a section at the water-grid borderline and gently removing the grid from the water. Subsequent drying was controlled and documented via the stereomicroscope. For electron tomography (ET), ribbons of multiple semithin sections (200-350 nm) were cut using an ultrasemi diamond knife (Diatome), stretched, and collected on grids. For optimal ribbon formation, the resin block was trimmed with a 20°diamond trim tool (Diatome) to provide parallel edges (Blumer et al., 2002) . To release ribbons from the knife, section thickness was reduced to 5 nm for 1-2 cutting movements. For SEM-BSD imaging, ultrathin sections were placed on freshly glow-discharged silicon wafers as substrates (Dittmayer et al., 2018; Grudniewska et al., 2018) .