Quantitative Assessment of Fluorescent Intensity

After imaging at 4080 × 3072 resolution, quantification of fluorescent intensity was performed via MetaMorph software (Molecular Devices) per a previously described protocol.¹⁴ Using high-resolution images at  ×2.5 magnification, measurements of fluorescent intensity (mean [SD] intensity and minimum and maximum intensities) were obtained from 5 regions of interest each from nerve proximal to the crush axotomy site, distal to the axotomy site, and surrounding background. Standardized sampling of regions of interest was conducted by placing the regions of interest at equidistant points from the crush injury longitudinally at the midpoint of the nerve t by an observer blinded to the corresponding experimental group. The mean value of the 5 points from each segment was then determined. A representative analysis is shown in Figure 1. Mean (SD) values were generated from these measurements and pooled for quantitative analysis.

A, Buccal branch imaged at × 2.5 magnification. The red circles in the fluorescent image represent regions of interest sampled to quantify fluorescence. A simplified systematic equidistant sampling along the length of each nerve site was performed to ensure representative sampling for quantitative analysis.