Dab substrate kit sk 4100

Surgical specimens of oligodendroglioma and diffuse astrocytoma were fixed in 4% paraformaldehyde and embedded in paraffin for routine histopathological and immunohistochemical analysis. Immunohistochemical analysis was performed using avidin-biotin immunoperoxidase technique. In brief, 4-μm-thick tissue sections were deparaffinized using xylene and rehydrated. After antigen retrieval using Target Retrieval Solution, pH 9.0 (Dako), the sections were autoclaved for 10 minutes and then maintained at room temperature for 40 minutes. Sections were quenched using 3% hydrogen peroxide (H₂O₂) in methanol for 20 minutes and blocked with 5% skim milk in TBST for 30 minutes. Sections were incubated with 3 μg/mL AMab-6 overnight at 4°C. Immunocomplexes were treated with an Envision+ Kit (Dako) for 1 hour. Color was developed using 3, 3-diaminobenzidine tetrahydrochloride (DAB Substrate Kit SK-4100, Vector, Japan) for 5 minutes. Sections were then counterstained with hematoxylin (Wako Pure Chemical Industries Ltd.). Images were acquired with a BZ-X700 microscope (Keyence, Osaka, Japan).

To retrieve the antigens, the sections were heated for 1 h at 90°C in citrate buffer (0.01 M, pH 6.0). After cooling, the sections were treated for 20 min with 0.3% hydrogen peroxide in distilled water to suppress the endogenous peroxidase activity. To avoid non-specific binding, the sections were treated with normal goat serum (Vectastain Elite ABC kit; Vector Laboratories, USA) for 1 h before being incubated overnight at 4°C with rabbit monoclonal anti-olfactory marker protein (OMP) (1:1,000 dilution, Cat. No. ab183947; Abcam, UK) and rabbit polyclonal anti-growth associated protein-43 (GAP-43) (1:1,000 dilution, Cat. No. PA5-34943; ThermoFisher Scientific, USA) antibodies. The primary antibodies were excluded from the procedure as negative controls. The sections were rinsed with phosphate-buffered saline (PBS), treated with biotinylated goat anti-rabbit IgG (Vectastain Elite ABC kit) for 1 h, and then reacted with the avidin-biotin-peroxidase complex (Vectastain Elite ABC kit) for 1 h at room temperature (RT). Immunoreactivity was detected using a diaminobenzidine substrate kit (DAB Substrate Kit SK-4100; Vector Laboratories), followed by counterstaining with hematoxylin.