Anti trpv1

Rat DRG, SC, and SSC were immediately excised at day 9 after behavior test for protein extraction. We followed the methods of Liao et al. 2016 [40 (link)]. Total protein was prepared by homogenizing the hippocampi for 1 h at 4°C in a lysis buffer containing 20 mmol/L imidazole-HCl (pH 6.8), 100 mmol/L KCl, 2 mmol/L MgCl₂, 20 mmol/L ethylene glycol tetraacetic acid (pH 7.0), 300 mmol/L sucrose, 1 mmol/L NaF, 1 mmol/L sodium vanadate, 1 mmol/L sodium molybdate, 0.2%  Triton X-100, and a proteinase inhibitor cocktail. From each sample, 30 μg of protein was extracted and analyzed through a bicinchoninic acid protein assay. The extracted protein was subjected to 10%–15%  sodium dodecyl sulfate-Tris-glycine gel electrophoresis and transferred onto a nitrocellulose membrane. The membrane was blocked with 5%  nonfat milk in a TBST buffer (10 mmol/L Tris-buffered saline, pH 7.5; 100 mmol/L NaCl; and 0.1%  Tween 20) and incubated overnight at 4°C with the anti-GABA_A antibody (1:1000, Alomone, Israel), anti-A1R antibody (1:1000, Alomone), anti-TRPV1 (1:1000, Alomone), anti-TRPV4 antibody (1:1000, Alomone), and anti-GluR3 antibody (1:1000, Alomone) in TBST containing bovine serum albumin. Peroxidase-conjugated antibody (1:500) was used as the secondary antibody. The membrane was assessed using the ECL-Plus protein detection kit.

Mice were anesthetized with 1% isoflurane and intracardially perfused, first with normal saline followed by 4% paraformaldehyde. Mice brains were then placed in 30% sucrose and embedded in tissue optimum cutting temperature (OCT)-freeze medium at –20°C on the following day. Frozen sections were cut (20 μm) and placed on amino propyltriethoxy silane (APS)-coated glass microslides. Subsequently, the sections were post-fixed in 4% paraformaldehyde for 3 min and incubated in blocking solution containing 3% bovine serum albumin (BSA, Merck, USA), 0.1% Triton X-100, and 0.02% NaN3 in phosphate buffered saline (PBS) for 2 h at room temperature. After blocking, brain sections were incubated with primary antibodies in a blocking solution at 4 °C overnight. The following primary antibodies were used: anti-TRPV1 (1:500, Alomone, Israel) and anti-pERK (1:500, Alomone, Israel) from Alomone. The secondary antibody was a goat anti-rabbit (1:500) antibody (MolecularProbes, Carlsbad, CA, USA). Slides were mounted with cover slips and visualized using a fluorescence microscope (CKX41 with an Olympus U-RFLT50 Power Supply Unit, Olympus, Tokyo, Japan).