Vectastain abc standard

Cells were fixed for 30 min with 4% paraformaldehyde, permeabilized for 10 min with methanol and treated for 10 min with 3% H₂O₂. After blocking of unspecific background with normal goat serum (1:10, DakoCytomation, Glostrup, Denmark) for 30 min, incubation with a mouse anti-PGP 9.5 antibody (1:1000, Acris, Herford, Germany) for 1 h was performed, followed by incubation with a biotinylated secondary antibody (goat anti-mouse IgG, 1:400) for 45 min and treatment with an avidin-biotin-complex (Vectastain ABC Standard, Vector Laboratories, Burlingame, CA, USA) conjugated with horseradish peroxidase for 45 min. Antibody binding was visualized with 3,3′-diaminobenzidine (DAKO, Hamburg, Germany).

Immunohistochemistry for the pan-neuronal marker PGP 9.5 was carried out as described previously [9 (link)]. Briefly, sections were incubated with 3% hydrogen peroxide, rinsed in TBS-buffer (TRIS-buffered saline; 10 mM TRIS, 50 mM NaCl, pH 7.4), incubated overnight with the polyclonal rabbit-anti-PGP 9.5 (PGP 9.5, 1:15000, Accurate Chemical & Scientific Corporation, Westbury, USA) diluted in antibody diluent (Invitrogen, Karlsruhe, Germany) and incubated for 45 min with biotinylated goat anti-rabbit IgG (1:400, DAKO, Hamburg, Germany). After washing three times with TBS, sections were incubated for 45 min with an avidin-biotin-complex (Vectastain ABC Standard, Vector Laboratories, Burlingame, USA) conjugated with horseradish peroxidase. 3, 3′-diaminobenzidine (DAKO, Hamburg, Germany) was used as substrate chromogen. Sections were counterstained with Meyer’s hematoxylin. Stained sections were analyzed by a light microscope (Nikon 6000, Nikon, Tokyo, Japan) coupled to a digital camera (Digital Sight, Nikon, Tokyo, Japan). Data acquisition was performed with NIS-Elements BR 3.2 software (Nikon, Tokyo, Japan). Omission of the primary or secondary antibody served as negative controls.