Rabbit anti tyrosine hydroxylase

Dewaxed sections and floating samples were treated with blocking of endogenous peroxidases (1% H₂O₂ in PBS), incubated in blocking solution [PBS + 0.2% bovine serum albumin (BSA)] for 1 h and then incubated overnight at 4°C with Rabbit Polyclonal Anti S100 (marker for myelinating and non-myelinating Schwann cells, Agilent-Dako, dilution 1:4,000), Rabbit Anti Tyrosine Hydroxylase (TH, for autonomic innervation, GeneTex, dilution 1:500), or Rabbit Anti PGP9.5 (as a pan-neuronal marker, Merck Millipore, dilution 1:500). After repeated PBS washing, the samples were incubated with the secondary Goat anti rabbit (Jackson, dilution 1:300) for 1 h. Negative controls were processed with the same protocol with the omission of the primary antibodies. The reaction was then developed with 3,3’-diaminobenzidine (Liquid DAB + substrate Chromogen System kit Dako) and stopped with distilled water. Samples were finally counterstained with hematoxylin.
The images were acquired by using Leica DMR microscope (Leica Microsystems, Wetzlar, Germany).

The floating fascia samples of thoracolumbar and gluteal region were fixed in formalin 10% in Phosphate Buffer Saline (PBS). After repeated washings in PBS, endogen peroxidases were blocked with 1% H₂O₂ in PBS for 20 min at room temperature. The floating tissues were then pre-incubated with a blocking buffer (BSA 0.1% in PBS) for 60 min at room temperature and then incubated in rabbit polyclonal Anti S100 (Dako, dilution 1:4000), Rabbit Anti Tyrosine Hydroxylase (TH, GeneTex, dilution diluited 1:700), Rabbit Anti PGP (GeneTex, dilution 1:500) in the same pre-incubation buffer and maintained overnight at 4 °C. We selected these antibodies because S100 is specific for Schwann cells forming myelin, TH stains the sympathetic nerve fibres, the anti-PGP is widely used as a marker for all peripheral nerve fibers. After repeated PBS washing, the floating thoracolumbar and gluteal fasciae were incubated for 1 h in goat anti rabbit HRP (Jackson ImmunoResearch—Laboratories, Inc., Cambridge -UK) diluted 1:300 in the same pre-incubation buffer and washed in PBS. Negative controls underwent the same protocol steps, with the omission of the primary antibodies. The reaction was then developed with 3,3′-diaminobenzidine (Liquid DAB + substrate Chromogen System kit Dako Corp, Carpinteria, CA, USA) and stopped with distilled water.