Qx 314

To block histamine receptors, antagonist of H1 receptor diphenhydramine (50 mg/kg, MilliporeSigma, St. Louis, MO) and antagonist of H4 receptor JNJ7777120 (30 mg/kg, MilliporeSigma, St. Louis, MO) were administered orally as a 20 min pretreatment in a volume of 300 μl sterile saline. Oral administration of 300 μl saline was used as control.
Flagellin (0.9 μg, MilliporeSigma, St. Louis, MO) was dissolved in 20 μl saline for intradermal injection on the skin behind the ears to evoke scratching responses. Flagellin (0.9 μg, MilliporeSigma, St. Louis, MO) and QX-314 (1%, MilliporeSigma, St. Louis, MO) were dissolved in 10 μl distilled PBS for intradermal injection to silence TLR5⁺ Aβ-LTMRs. QX-314 (1%) was dissolved in 10 μl distilled PBS as control.
For formalin test, 50 ul of formalin (5%, MilliporeSigma, St. Louis, MO) was injected into the plantar region of the hindpaw and licking, biting or flinching was recorded for an hour.

WS-12, QX-314, allyl isothiocyanate, cinnamaldehyde, and capsaicin were purchased from Sigma Aldrich. Artemin was purchased from R&D Systems, CGRP(8–37) receptor antagonist from Tocris, TRPM8 antagonist PBMC from Focus Biomolecules, and TLR4 antagonist TAK-242 from Calbiochem.
Ethanol was used as vehicle throughout this study except for injections of CGRP(8–37), TAK-242 and Artemin in which 0.9% saline was used as the vehicle. All injections were at a volume of 20μl and intraplantar in one hind paw for all experiments, except for Cold Plate where both fore paws were injected with 10μl, as previously described [30 (link); 39 (link)]. Amounts injected of each chemical were: cinnamaldehyde 120ng and 50μg, capsaicin 1μg, allyl isothiocyanate 0.15%, CGRP(8–37) 6.4mmol, TAK-242 0.2μg, WS-12 20μg, PBMC 100μg, Artemin 0.2μg, and QX-314 1%.

Saline, lidocaine (Sigma-Aldrich), flagellin (InvivoGen, San Diego, CA), and QX-314 (Sigma-Aldrich) were microinjected into the right L5 DRG using a previously described technique [11 (link),20 (link)]. Briefly, the intervertebral foramen was exposed surgically and then minimal foraminotomy was performed to expose the dorsal aspect of the distal pole of the DRG. Injection was performed with a microinjector (Nanoliter 2000, World Precision Instruments, Sarasota, FL, USA). A glass micropipette filled with solution was advanced ~100μm into the ganglion, followed by injection of 3μl of solution with drugs, a volume that has been shown to fully fill the rat L4 and L5 DRG [20 (link)]. Injection was performed over a 5-min period and removal of the pipette was delayed for an additional 5min to minimize the extrusion of the injectate. Overlying muscle and skin were closed. Rats fully recovered from anesthetics in 30 minutes after the surgery and examination of evoked behaviors were initiated 45min after the injection.

Picrotoxin and QX-314 were purchased from Sigma. LY379268, LY395756, and LY341495 were purchased from Tocris Bioscience. VU6001192 and VU0469942 were generously provided by the Vanderbilt Center for Neuroscience Drug Discovery (Vanderbilt University, Nashville, TN).

Propofol was purchased from APP Pharmaceuticals, LLC (Schaumburg, IL). Corticosterone, TTX, and QX314 were acquired from Sigma-Aldrich (St. Louis, MO). Bumetanide (Ben Venue Laboratories, Inc., Bedford, OH) was purchased from Bedford Laboratories (Bedford, OH). AP5 and DNQX were purchased from Tocris Cookson, Inc. (Ellisville, MO).