Histostain plus

Paraffin-embedded colonic sections were deparaffinized in xylene and rehydrated in graded alcohol. After quenching the endogenous peroxidase and blocking the nonspecific binding, sections were incubated with mouse monoclonal antibody against NF-κB p65 subunit at 1 : 100 dilution or rabbit polyclonal antibody against IL-1β at 1 : 50 dilution overnight at 4°C. Sections were then incubated with biotinylated secondary antibody at room temperature for 20 min, incubated with avidin-biotin complex reagent, and stained with 3,3′-diaminobenzidine (Histostain-Plus, Invitrogen, Camarillo, CA, USA).

Cross-sections 2 μm in thickness were taken with a microtome (Leica MR 2145) from paraformaldehyde-fixed paraffin-embedded eye tissues, floated in a sterile bath, placed onto poly-L-Lysine-coated glass slides, and dried at room temperature. After overnight incubation at 60 °C, the slides were dewaxed in xylene for 30 min, rehydrated through a graded ethanol series (100%, 95%, 80%, and 70%, sequentially), washed in distilled H₂O and PBS for 10 min, treated with 2% trypsin containing 50 mM Tris buffer (pH 7.5) at 37 °C for 15 min, and then washed again with PBS. Sections were delineated with a Dako pen (Dako, Glostrup, Denmark), incubated in 3% H₂O₂ solution for 15 min to inhibit endogenous peroxidase activity, and washed with PBS. The slides were incubated with VEGF primary antibody at 57 °C followed by washing with PBS. Afterwards, a biotinylated secondary IgG antibody was applied and washed with PBS before incubating with the streptavidin-peroxidase conjugate (Histostain Plus, Invitrogen, Camarillo, CA, USA) for 30 min to visualize the immunostaining. The whole procedure was finished after counterstaining the sections with Mayer’s hematoxylin (Sigma Chemical Co., St. Louis, MO, USA). All sections were examined and photographed with an Olympus C-5050 digital camera mounted on an Olympus BX51 microscope (Olympus Corp., Tokyo, Japan).

The tissue blocks containing the most representative areas from 14 radical prostatectomy specimens (containing normal, high grade prostatic intraepithelial neoplasia [H-PIN] and adenocarcinoma) were chosen based on H&E staining, and 5 µm sections were cut and mounted on poly-L-lysine-coated slides for immunohistochemical staining. A standard streptavidin-biotin immunoperoxidase method was used to stain the tissue sections with ß-catenin and NKX3.1 antibodies (Santa Cruz Biotechnology, dilution 1/200). Briefly, the tissue sections were treated in xylene, rehydrated in an alcohol series and immersed in distilled water. Endogenous peroxidase activity was blocked using a 0.3% solution of hydrogen peroxide in phosphate-buffered saline at room temperature (RT) for 10 min, and the sections were then rinsed with washing buffer (50 mM Tris-Cl, pH 7.5). The primary antibodies were applied for 1 h at RT, the sections were washed, the streptavidin-labeled peroxidase-conjugated antibody (Invitrogen, Histostain Plus, 85–9043) was added at RT for 10 min, and the sections were washed again. The peroxidase activity was visualized with 0.03% 3,3-diaminobenzidine tetrahydrochloride (DAB) (Sigma Chemical Co., St. Louis, Missouri, USA) for 5 min. The sections were then washed in deionized water, counterstained with Mayer's hematoxylin and mounted.