Biotinylated goat anti rabbit igg

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.

The apoptosis rate of insulin cells was determined by double immunocytochemical labelling for active Caspase-3 and insulin.
To determine insulin-positive cells, the biotinylated-streptavidin-peroxidase immunocytochemical method was carried out. For this, sections were incubated with monoclonal mouse anti-insulin (diluted 1:1000 in TBS, Sigma) overnight at 4°C, followed by incubation with biotinylated goat anti-mouse IgG (Dako, diluted 1:100, in TBS) and streptavidin-horseradish peroxidase complex (Dako, diluted 1: 100 in TBS), applied successively at room temperature for 40 min each. The reactions were developed in freshly prepared 3–3'DAB (0.025% in TRIS buffer containing 0.03% of H₂0₂). The second reaction for active caspase-3 was visualized by immunofluorescence. The sections were incubated with polyclonal rabbit anti-active caspase-3 (Sigma, diluted 1: 500 in TBS) overnight at 4°C, followed by incubation with biotinylated goat anti-rabbit IgG (Dako, diluted 1:100, in TBS) and Streptavidin-Cy3 complex (Sigma, diluted 1: 100 in TBS) applied successively at room temperature for 40 and 60 min, respectively.

Immunohistochemical staining (using the streptavidin–biotin–peroxidase method) was performed with mouse monoclonal antibody against human β-catenin (1:100 dilution; Cell Signaling #8480, Massachusetts, USA), and biotinylated goat anti-rabbit IgG (Dako, Tokyo, Japan). The staining intensities and sites of β-catenin in the endometriotic cysts of the ovaries compared with normal endometrial tissue were assessed.