Qwin image analysis software package

At 21 days post surgery, animals in five groups were transcardially perfused with 4% paraformaldehyde in PBS. The distal portion of crushed and contralateral, uncrushed sciatic nerves were harvested. The nerve sample was fixed with 2.5% glutaraldehyde, post-fixed with 1% osmium tetraoxide solution at 4°C, dehydrated stepwise in increasing concentrations of ethanol, embedded in Epon 812 epoxy resin, and cut into transverse sections. The semi-thin section was stained with toluidine blue for measuring the cross-sectional area of the total nerve trunk area under light microscopy, and ultra-thin sections were stained with lead citrate and uranyl acetate for examination under transmission electron microscopy (JEOL Ltd., MA, USA). The electron micrographs from six random fields (5,000 × magnification) of each nerve section were digitalized into a Q550 IW image analysis system (Leica Imaging Systems Ltd.) and analyzed with a Leica Qwin image analysis software package to obtain morphometric parameters, including myelin thickness and diameter of myelinated axons.

Bilateral gastrocnemius muscles were harvested from animals post-sacrifice. The muscle sample from the mid-belly was post-fixed in buffered 4% paraformaldehyde for 24 hours, dehydrated in a graded ethanol series, cleared in xylene, embedded in paraffin and cut into 5-μm-thick transverse sections, which were processed with hematoxylin and eosin (HE) staining. Stained sections were photographed with a DC 300F color digital camera, and digitalized into a Q550 IW image analysis system (Leica Imaging System Ltd.). Six visual fields were selected randomly for each specimen and the cross-sectional areas (CSA) of the gastrocnemius muscle fibers were measured by the aid of Leica Qwin Image Analysis software package.