Rabbit anti trpv1 antibody

The spinal cord was lysed in ice-cold RIPA buffer with a protease inhibitor cocktail (Sigma Aldrich, St. Louis, MO). Tissues were homogenized using a tissue homogenizer. Spinal cord samples were loaded into the 10% polyacrylamide gels and then were electrotransferred onto a nitrocellulose membrane. The membranes were blocked with 10% non-fat dry milk in PBS with 0.3% Tween for 1 hour and then incubated overnight at 4°C with rabbit anti-TRPV1 antibody (1:500; Alomone Labs, Jerusalem, Israel), or GAPDH antibody (1:5000; Sigma Aldrich, St. Louis, MO) diluted in PBS. The membrane was then incubated in horseradish peroxidase-conjugated anti-rabbit IgG (1:5000; Santa Cruz Biotechnology, Dallas, TX) for 2 hours at room temperature. The antigen–antibody complexes were visualized using an enhanced chemiluminescence detection reagent (Bio-Rad Laboratories, Hercules, CA). Bands were scanned using a densitometer (GS-700; Bio-Rad Laboratories, Hercules, CA), and quantification was performed using NIH Image J software.

The ECs were grown on coverslips in Petri dishes with M199 medium (Sigma-Aldrich St Louis MO) supplemented with 10% FBS (HyClone Thermo-fisher Waltham Mass USA) and an antibiotic-antimycotic mixture (Sigma-Aldrich St Louis MO) for 48 hrs. at 37°C in an atmosphere with 5% CO2. Subsequently, cells were stimulated with CS for 30 minutes. Then, the cells were fixed with a 4% paraformaldehyde solution for 10 minutes at 4°C. The cells were permeabilized with Triton X-100 0.1% (Sigma-Aldrich St Louis MO) at room temperature for 10 minutes, followed by incubation with a rabbit anti-TRPV1 antibody 1 : 50 (Alomone Labs Jerusalem ISR), in a humidified chamber overnight at 4°C. An unlabeled anti-TRPV1 antibody was detected by incubation with an anti-rabbit anti-IGg antibody conjugated with fluorescein isothiocyanate (FITC) (Thermo Fisher Waltham Mass USA). The cells were examined by confocal fluorescence microscopy with an inverted LSM 700 Axio Observer confocal microscope (Carl Zeiss Jena GmbH) at a 40X magnification. We include a 2.5 X digital zoom image in order to appreciate more cell details. DAPI was used to contra-stain the nucleus. The intensity of fluorescence was determined by the zen blue program (Carl Zeiss Jena GmbH).