Polyclonal rabbit anti rat

L4–6 spinal cord tissues were collected from each group of animals. The total protein of the ipsilateral spinal cords was extracted using radioimmunoprecipitation assay protein lysis buffer (Beyotime Institute of Biotechnology). Protein concentration was determined using a bicinchoninic acid assay kit (Beyotime Institute of Biotechnology). Equal quantities of protein (200 µg) were fractionated using SDS-PAGE (10%; Beyotime Institute of Biotechnology), transferred onto polyvinylidene difluoride membranes (Merck Millipore, Darmstadt, Germany) and then blocked with 10% non-fat dry milk for 3 h at room temperature. Primary antibodies for c-fos (cat. no. sc-52), TRPV1 (cat. no. sc-28759; polyclonal rabbit anti-rat; 1:1,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and GAPDH (monoclonal rabbit anti-rat; 1:10,000; Epitomics, Burlingame, CA, USA) were incubated overnight at 4°C, followed by probing for 2 h at room temperature with an anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:5,000; Santa Cruz Biotechnology, Inc.). Finally, the immunoreactive bands were visualized in enhanced chemiluminescence solution (Pierce Biotechnology, Inc., Rockford, IL, USA) and exposed onto X-ray films.

Immunoblotting analysis was used to identify NOS I and NOS III. Aliquots of samples containing 50 μg of proteins were loaded on 7.5% SDS polyacrylamide gels and then blotted on to PVDF membranes (GE Healthcare, Amersham Hybond‐P) at 60 V for 3 h. The membranes were washed in TBST buffer (50 mmol/l Tris‐ based saline, pH 7.4, containing 0.1% Tween 20) and blocked with 7,5% skimmed milk in TBST for 1 h. Blots were incubated overnight at 4°C with the NOS I antibody (polyclonal rabbit anti‐rat; diluted 1:500; Santa Cruz Biotechnology, Dallas, Texas), which recognized a 155‐kDa band, or with the NOS III antibody (polyclonal rabbit anti‐rat; diluted 1:250; BD Transduction Laboratories, San Jose, California) which recognized a 140‐kDa band. Beta‐tubulin was used as loading control (rabbit anti‐rat beta‐tubulin, 50‐kDa band; Abcam Inc., Cambridge, MA). The membranes were then incubated with a donkey anti‐rabbit IgG horseradish peroxidase‐conjugated secondary antibody (1:3000) (Abcam Inc., Cambridge, MA). Blots were visualized using a quimioluminiscent substrate Bio‐Lumina (from Kalium Technologies, BA, Argentina).
The relative protein levels were determined by analyzing the bands with Gel Pro Analyzer 3.1 for Windows and relative protein expression was calculated as the ratio NOS I (or NOS III)/β‐tubulin.