Monoclonal goat antibody against th

Two months after the therapeutic intervention, animals were euthanized with an overdose of pentobarbital sodium (80 mg/kg) and an intracardiac perfusion with 4% paraformaldehyde was performed. The affected portions of the SC were fixed overnight and then transferred to 30% sucrose for cryoprotection. Samples were embedded in Tissue-Tek (Miles Elkhart, IN, USA), and longitudinal frozen sections (40 μm thick) were performed. Immunohistochemical staining was carried out in order to count the amount of TH+ and 5-TH+ fibers. Tissues were incubated in 0.03% hydrogen peroxide to quench endogenous peroxidase activity. Subsequently, the tissue was incubated overnight with the following primary antibodies: monoclonal goat antibody against TH (1:2000; Chemicon), or polyclonal rabbit antibody against 5-HT (1:2000; Sigma- Aldrich). Following rinsing with PBS, samples were incubated for at least 2 h with donkey IgG anti-goat IgG (1:500; Chemicon) and Sheep IgG anti rabbit IgG (1:500; Abcam) secondary biotinylated antibodies. To visualize positive fibers, samples were incubated 5 min with Vector DAB kit (Vector laboratories, CA, USA). Then, samples were evaluated and analyzed by a blinded observer that counted individual fibers using a 20X objective (Olympus DP72, Japan). The number of regenerating axons 1 mm caudal to the lesion was assessed.

Eight weeks after the therapeutic intervention, animals were euthanized with an overdose of sodium pentobarbital (80 mg/kg) and intracardiac perfusion with 4% paraformaldehyde. The affected portions of the SC were fixed overnight and then transferred to 30% sucrose for cryoprotection. Samples were embedded in Tissue-Tek (Miles Elkhart, IN, USA), and longitudinal frozen sections (40 μm thick) were performed. Immunohistochemical staining was carried out in order to count the amount of positive TH and 5-TH fibers. Tissues were incubated in 0.03% hydrogen peroxide to quench endogenous peroxidase activity. Subsequently, the tissue was incubated overnight with the following primary antibodies: monoclonal goat antibody against TH (1:2000; Chemicon), or polyclonal rabbit antibody against 5-HT (1:2000; Sigma-Aldrich). Following rinsing with PBS, samples were incubated for at least 2 h with donkey IgG anti-goat IgG (1:500; Chemicon) and Sheep IgG anti rabbit IgG (1:500; Abcam) secondary biotinylated antibodies. To visualize positive fibers, samples were incubated 5 min with Vector DAB kit (Vector laboratories, CA, USA). Then, samples were evaluated and analyzed by a blinded observer that counted individual fibers using a 20× objective (Olympus DP72, Japan). The amount of regenerating axons at the epicenter and 1 mm caudal to the lesion was assessed.