Trpv1

The temporal bones were removed and fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 1.5 hr at 4°C. The temporal bones were decalcified by incubation in 10% EDTA at 4°C for 3–5 days. The EDTA solution was changed daily. The bones were then embedded in the OCT compound for cryostat sectioning. The sections of 10 μm thickness were washed in PBS, and nonspecific binding was blocked with 1% bovine serum albumin (BSA) and 10% goat serum in PBS plus 0.1% Triton X-100 (PBST) for 1 hr. The primary antibodies, chicken anti-Tuj1 (Abcam), mouse anti-myelin basic protein (Abcam), rabbit anti-Myo7a (Proteus Biosciences, Inc), mouse anti-Tuj1 (Abcam), rabbit anti-TRPA1 (Abcam), TRPC3 (Novus Biologicals), TRPC6 (Abcam), TRPV1 (Novus Biologicals), TRPV4 (Abcam), were incubated overnight at 4°C. After incubating the primary antibodies, the slides were washed three times with PBST and incubated with secondary antibodies for 1.5 hr at RT in the dark. We used Alexa Fluor 647-conjugated goat anti-mouse and Cy3-conjugated goat anti-chicken, Alexa Fluor 488-conjugated goat anti-rabbit, Alexa Fluor 568-conjugated goat anti-mouse (Jackson ImmunoResearch Labs) in a dilution of 1:500. Other markers used were phalloidin-Fluor 647 (Abcam) for F-actin and DAPI (Sigma) for nuclear stain. The slides were then examined under a confocal microscope (LSM 510, Zeiss).

Western blots of spinal cord dorsal horn sections with TLR4 (1: 1000, Proteintech, Rosemont, IL, USA, 19188-1-AP), MyD88 (1:1000 Proteintech, 23230-1-AP), p-NF-κB (1:1000, cell signaling, 3033), NF-κB (1:1000, cell signaling, 8482) and TRPV1 (1: 1000; Novus Biologicals, Centennial, CO, USA, NBP1-97417) were produced. The protein bands in the blot were visualized using the ECL Western Blotting Detection Kit and Bio-Rad ChemiDoc XRS system. The band intensity was quantified and plotted using Quantity One Software (Version 4.6.8, Bio-Rad, Hercules, CA, USA).