Identical tissue sections for both analysis procedures were used to eliminate potential fluorescent intensity variance between slides. Additionally, FITC-conjugated secondary antibody was examined as well as Rhodamine-conjugated secondary antibody to identify whether intrinsic differences between different fluorophores (red vs. green) could yield false-positive group differences. Inhibitors,research,lifescience,medical IL-1β IR was examined with standard FITC fluorescent analysis utilizing NIH Image J software procedures (Fig. 3A). While a trend toward differences was present using Image J

software, no statistically significant increase in IL-1β IR in either the ipsilateral or contralateral dorsal horn was found between non-neuropathic (Sham-Vehicle) and neuropathic CCI-treated rats (Student’s t test P = 0.0620 and P = Inhibitors,research,lifescience,medical 0.5142, respectively). We utilized FITC-tagged secondary antibody in these studies because FITC tends to fade at a greater rate than Rhodamine Red,

providing a stringent assessment of potential observed differences between experimental groups following a subsequent exposure. We therefore exposed the same tissue sections analyzed in Figure 3A Inhibitors,research,lifescience,medical for a second time (Fig. 3B) with double the CAL-101 molecular weight exposure duration, but with the light sensitivity held consistent. Doubling the exposure time provides a rigorous test to determine whether fading can influence quantitative results. In using Image J, marginal nonsignificant fading of fluorescent intensity (Fig. 3A vs. 3B) (Student’s t test P = 0.7418 and P = 0.9060, respectively) was present, and no difference in IL-1β IR between non-neuropathic and neuropathic rats was detected (Student’s t test Inhibitors,research,lifescience,medical P = 0.0648 and P = 0.4874, respectively). In the same context, marginal fading was observed with spectral microscopy Inhibitors,research,lifescience,medical exposure between an initial exposure and a subsequent exposure (data not

shown). Given that fluorophore fading was not present upon subsequent exposures, a new set of FITC-stained IL-1β tissues from nonneuropathic (Sham-Vehicle) and neuropathic rats (CCI-Vehicle) treatment groups were examined with standard Image J fluorescence analysis followed by spectral analysis. With Image J, we found no significant effect of surgery in either ipsilateral or contralateral dorsal horn (Student t test P = 0.5604 and P = 0.6988, respectively) (Fig. 3C). However, spectral analysis of the identical sections revealed either statistically significant differences in ipsilateral IL-1β IR due to surgical treatment (Student’s t test P = 0.0482 and P = 0.0635, respectively) (Fig. 3D). We found similar effects following comparison with a new set of slides from L4–L6 lumbar spinal cord tissue sections treated with IL-1β primary antibody, but incubated with a secondary antibody conjugated to the Rhodamine Red fluorophore (Fig. 3E and 3F).