Diaminobenzidine substrate kit

The SN tissues from the mice were fixed with 4% PFA for 48 h, immersed in 30% (w/v) sucrose solution, and sectioned at a thickness of 30 μm. The free-floating sections were incubated with 1% (v/v) H₂O₂ in 0.5 M PBS for 15 min, blocked with 5% (v/v) normal goat serum (NGS) for 1 h, and incubated with anti-tyrosine hydroxylase (TH) primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4 °C. The sections were incubated with Vectastain Elite ABC reagents (Vector) for 1.5 h at RT, incubated with Diaminobenzidine Substrate Kit (Vector) for 10 min at RT, and then mounted on gelatin-coated slides. Images of TH-positive cells in the SN were captured using an Axio Scope A1 microscope and AxioCam ICc3 camera (Zeiss). The TH-positive cells were counted on each capture and confirmed three times.

Immunohistochemical staining was performed on 5-μm paraffin sections. After the sections were deparaffinized, heat-induced epitope retrieval was performed by immersing the slides in 0.01 M sodium citrate buffer (pH 6.0). To block endogenous peroxidase activity and the nonspecific binding of antibodies, the sections were preincubated for 1 h at room temperature in 0.1 M phosphate-buffered saline containing 10% normal goat serum and 0.3% H2O2. The sections were incubated with one of two primary antibodies: rabbit polyclonal anti-von Willebrand factor (vWF) antibody (1:100; Abcam, Cambridge, MA, USA) or rabbit polyclonal anti-vascular endothelial factor (VEGF) antibody (1:50; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The sections were treated with biotinylated goat antirabbit immunoglobulin G (IgG; 1:200, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). An avidin–biotin complex kit (Vector Laboratories, Inc., Burlingame, CA, USA) and a diaminobenzidine substrate kit (Vector Laboratories, Inc.) were used to visualize the brown reaction product, following the manufacturer’s recommendations. Vascular density was determined through immunohistochemical staining for vWF. Microvessel density was determined by counting the number of vessels positive for vWF staining in at least four random lung fields at 400× magnification in an unbiased manner [20 (link)].

Tumors were fixed by formalin and paraffin embedded tissue blocks were dewaxed, rehydrated, and blocked for endogenous peroxidase activity. Antigen retrieving was performed in sodium citrate buffer (0.01 mol per Litter, pH 6.0) in a microwave oven at 1000 W for 3 min and then at 100 W for 20 min. Nonspecific antibody binding was blocked by incubating with 10% fetal bovine serum in PBS for 30 min at room temperature. Slides were then incubated with anti-Ki67 (at 1:500; Neomarker), anti-AR (at 1:200; Santa Cruz Biotechnology) or anti-AR-V7 (at 1:200; Precision) at 4 °C overnight. Slides were then washed and incubated with biotin-conjugated secondary antibodies for 30 min, followed by incubation with avidin DH-biotinylated horseradish peroxidase complex for 30 min (Vectastain ABC Elite Kit, Vector Laboratories). The sections were developed with the diaminobenzidine substrate kit (Vector Laboratories) and counterstained with hematoxylin. Nuclear staining of cells was scored and counted in 5 different vision fields. Images were taken with an Olympus BX51 microscope equipped with DP72 camera.

Nek6 protein expression in the clinical specimens of HCC and non-HCC tissues was determined by immunohistochemistry. The formalin-fixed samples were paraffin-embedded and sectioned (4-μm). The slides were incubated with rabbit anti-human Nek6 polyclonal antibody (1:100 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at 37°C for 2 h, where the normal rabbit IgG1 monoclonal antibody (1:100; Santa Cruz Biotechnology, Inc.) was used as a negative control (NC). This was followed by incubation with a horseradish peroxidase-conjugated goat anti-rabbit secondary monoclonal antibody (Dako Japan Co., Ltd., Kyoto, Japan) at 37°C for 1 h. The signals were detected using the diaminobenzidine substrate kit (Vector Laboratories, Burlingame, CA, USA) and counterstaining was performed with hematoxylin.

Immunohistochemical staining was performed on 5-μm paraffin sections. After the paraffin sections were deparaffinized, heat-induced epitope retrieval was performed by immersing the sections in 0.01 M sodium citrate buffer (pH 6.0). To block endogenous peroxidase activity and the nonspecific binding of antibodies, the sections were preincubated for 1 h at room temperature in 0.1 M phosphate-buffered saline containing 10% normal goat serum and 0.3% H₂O₂. The sections were incubated with rabbit polyclonal anti-vWF antibodies (1:200, GTX60934; GeneTex, Irvine, CA, USA) for 20 h at 4 °C and biotinylated goat anti-rabbit IgG (1:200, Jackson ImmunoResesarch Laboratories, West Grove, PA, USA) for 1 h at 37 °C. The avidin–biotin complex kit (Vector Laboratories, Newark, CA, USA) and diaminobenzidine substrate kit (Vector Laboratories) were used to visualize the brown reaction product according to the manufacturer’s recommendations. All immunostained sections were viewed and photographed using an Olympus BX43 microscope. Digital images of each section were captured and quantified by counting the positive stained vessels in five randomly selected fields per section at × 400 magnification.