Mglur5

Rats were transcardially perfused first with 250mL of chilled PBS, then 300mL of 4% paraformaldehyde (PFA). The C6/C7 spinal cord was dissected out, post-fixed overnight in 4% PFA, and then submerged in 30% sucrose solution for 5-7 days for cryoprotection. Spinal cords were embedded in OCT medium and frozen, and then 14μm cryosections were mounted on Fisher Superfrost slides. Sections were blocked for 2 hours at room temperature with 10% normal goat serum with 0.3% Triton-X, and then labeled overnight at 4°C with primary antibodies for pNR1 (rabbit, 1:500; Abcam; Cambridge, MA), mGluR5 (rabbit, 1:1000; Millipore; Billerica, MA), or GFAP (mouse, 1:500; Millipore; Billerica, MA). Slides were rinsed with PBS and labeled with goat anti-mouse Alexa 488 and goat anti-rabbit Alexa 568 secondary antibodies, then coverslips were mounted with Fluorogel medium (Electron Microscopy Sciences; Hatfield, PA). The spinal dorsal horns were imaged at 200x using an Olympus BX51 microscope, and analyzed using densitometry techniques in a customized MATLAB code to quantify the percentage of pNR1-, mGluR5-, or GFAP-positive pixels in each image [16 (link),30 (link)]. The percentage of positive pixels in the injury groups with vehicle or bupivacaine treatment were normalized to sham values and compared by one-way ANOVA with post-hoc Tukey's HSD test.

Equal aliquots from each fraction separated by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked for 1 hr in 5% milk/Tris-buffered saline and probed overnight at 4°C with the following primary antibodies diluted in 5% milk/Tris-buffered saline containing 0.1% Tween-20: calnexin (Enzo Life Sciences, Farmingdale, NY, USA), syntaxin1a (Abcam, Cambridge, MA, USA), mGluR5, PKCε, phospho-(Ser729)-PKCε (all from Millipore, Billerica, MA, USA) and ERK1/2 and phospho-ERK1/2 (both from Cell Signaling Technology, Danvers, MA, USA). After incubation with an appropriate horseradish peroxidase-conjugated secondary antiserum (Jackson ImmunoResearch, West Grove, PA, USA), immunoreactive bands on the membranes were detected by ECL+ chemiluminescence reagents on an X-ray film (GE Healthcare, Piscataway, NJ, USA). Subsequently, blots were stripped and re-probed with calnexin and syntaxin-1a antibodies to monitor biotinylation of intracellular proteins and to normalize for unequal loading and/or transfer of proteins. The integrated band density of each protein sample was measured using NIH Image J software version 1.32j (RRID: SCR_003070). All primary antibodies used in this study are described in detail in Table 1.