After washing with the buffer solution and post-fixation in 1% OsO4 and 0.1 m cacodylate buffer (pH 7.4), they were washed with the buffer solution, dehydrated using alcohol and acetone, and embedded in epoxy resin.
Both CCAs were removed and washed in phosphate buffer solution, dehydrated on dry ice, and stored at −80°C for 24 h.
Samples with the most frequent failure mode of each group were selected, fixed in 2.5% glutaraldehyde in cacodylate buffer solution, dehydrated in ascending concentrations of ethanol, and chemically dried using hexamethyldisilazane.
The samples were gently washed with phosphate buffer solution and then dehydrated by successive passages through 50%, 70 %, 80 %, 90 and 100% ethandl.
Proximal and distal segments of the nerves were postfixed in 1% OsO4 in cacodylate buffer solution and dehydrated in graded ethanol.
All specimens were fixed in 2.5% glutaraldehyde in cacodylate buffer solution and dehydrated in ascending concentrations of ethanol prior to embedding in epoxy resin (Agar Scientific, Essex, UK), after which ultra-thin sections were cut (Ultracut UCT, Leica, Vienna, Austria).
After washing with PIPES buffer, the samples were stained with 1% osmium tetroxide (OsO4) (Sigma Aldrich, USA), washed twice with PIPES buffer and dehydrated by immersing in an ethanol series.
The gel pieces were then washed with 100 mM NH4HCO3 buffer and dehydrated by ACN several times repeatedly and dried in a vacuum centrifuge.
Cells were washed with buffer and dehydrated by using a graded series of ethanol rinses (33, 50, 75, 90, and 3 × 100% ethanol).
Then the postfixed tissues were washed in same buffer and dehydrated by graded series of ethanol starting at 50% each step for 10 min and finally with propyleneoxide.
