*5.9. Nerve Growth Factor Expression by Western Blotting Assay*

On the 17th day after immunization, animals were euthanized under anesthesia and the L3-L6 regions of the spinal cord were removed. The samples were homogenized in buffer containing Hepes-NaOH (1M, pH 7.9), EGTA (200 mM), NaCl (0.9%), Triton-X 100 (1%), and phosphatase and protease inhibitor (1:300, Sigma-Aldrich™, USA). The samples were centrifuged at 12,000× *g* rpm for 20 min at 4 ◦C. After centrifugation, an aliquot of the supernatant was used for protein concentration determination using the method of Bradford (Thermo Fisher Scientific, Waltham, MA, USA) [101]. Aliquots containing 30 μg of protein were boiled in Laemmli buffer 5× and after that subjected to polyacrylamide gel electrophoresis (SDS-PAGE), in an Mini-Protean apparatus for mini gel (BioRad, Hercules, CA, USA). After separation by electrophoresis, the proteins were transferred to a nitrocellulose membrane (BioRad, Hercules, CA, USA), and subsequently, the membrane was blocked in TBST (20 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween 20) containing 5% of BSA for 1 h, then washed in TBST, followed by incubation with antibody NGF (1:1000, Abcam, Boston, MA, USA) overnight at 4 ◦C. Then, membranes were incubated with anti-IgG from the animal producing the respective peroxidase-conjugated primary antibody (1:5000, Abcam, USA), for 2 h at room temperature. ECL (Pierce) solution with a digital image capture system (UVITEC Cambridge) was used for bands visualization and the UVITEC Cambridge program was used for optical density determination. Results were normalized by membrane incubation with beta-actin (1:5000, Sigma Aldrich™, USA).

#### *5.10. Transmission Electronic Microscopy*

After intraperitoneal anesthesia with urethane (3 g/kg), animals were perfused with modified Karnovsky fixative solution containing 2.5% glutaraldehyde and 2% PFA in 0.1M sodium phosphate buffer solution (pH 7.4) [102]. After dissection of the muscles of the posterior thigh, the sciatic nerve was collected. The samples were post-fixed in 1% osmium tetroxide solution at 4 ◦C and then immersed in 5% aqueous uranyl acetate solution at room temperature. After that, the samples were dehydrated using a series of alcohols, then immersed in propylene oxide, and finally included in Spurr resin. Semi-thin sections (15 μM) were cut using Reichert Ultra Cut® ultra-microtome (Leica, Wetzlar, Germany) and stained with 1% toluidine blue solution. Subsequently, the 60 nm ultrathin sections were collected on 200 mesh copper grids (Sigma-Aldrich™, USA) and contrasted with 4% uranyl acetate solution and 0.4% aqueous lead citrate solution [103]. The grids were examined using the Jeol 1010 transmission electron microscope (NEP/MEPA/ESALQ) at the University of São Paulo.
