Liquid dab substrate chromogen system kit

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).

For immunohistochemistry, the slides were immersed in an antigen retrieval solution consisting of EDTA/Tris, pH 9.0, at 125 °C in an electric pressure cooker managed by a REPTIDE microprocessor (Celerus). Nonspecific binding of the antibodies was blocked by incubation of the slide with hydrogen peroxide. Next, the slides were incubated with the primary ADPH antibody (clone AA5-27, LifeSpan) diluted 1/100. To amplify the antigen–antibody binding, two other antibodies were applied to the slides, post-primary (Novolink polymer Novocastra) and the polymer (Novolink polymer Novocastra). The reaction was developed with diaminobenzidine (DAB) as a chromogen (Liquid DAB + Substrate Chromogen System Kit, DakoCytomation, Carpinteria, CA, USA).