Leitz laborlux s

Nerves from Lewis rats, including the regenerating tissue from the proximal nerve end or ANA, were harvested and marked with a proximal suture, then stored in 3% glutaraldehyde (Electron Microscopy Sciences; Hatfield, PA) in 0.1 M pH 7.2 phosphate buffer (Fisher Scientific; Fair Lawn, NJ) at 4° C until processing. The explanted tissues were processed and analyzed as described previously^{32 (link)}. Briefly, the tissues were post-fixed in 1% osmium tetroxide (Polysciences, Inc., Warrington, PA), and serially dehydrated in 90% ethanol (Thermo Fischer Scientific, Winmill Hill, UK). The tissues were then embedded in grade 502 epoxy (Polysciences, Inc., Warrington, PA) and sectioned to 1 μm with an ultramicrotome. The slides were stained with 1% toluidine blue and analyzed under light microscopy at 1000x (Leitz Laborlux S, Leica; Buffalo Grove, IL) using the IA32 Image Analysis System (Leco; St. Joseph, MI) in order to quantify nerve fiber counts, fiber width, fiber density, and percent neural tissue. One hundred twenty (120) days after the initial nerve injury, the number of myelinated axons within the proximal, middle and distal segment of regenerated tissue, as well as proximal to the initial injury were examined. All analysis was conducted by a blinded observer to the experimental groups.