Anti il 6

A non-biotin-amplified method (NovoLinkTM Polymer Detection System, Novocastra Laboratories, Newcastle Upon Tyne, UK) was used for visualizing antigens in tissue sections. The sections were subsequently incubated with primary anti-E-cadherin (clone 32A8, Cell Signaling Technology, Beverly, MA, USA), anti-IL-6 (Sigma-Aldrich) and anti-p53 (Novocastra) antibodies diluted in 1% bovine serum albumin in Tris buffer saline overnight at 4 °C using the appropriate dilutions. The slices were then counterstained with hematoxylin, dehydrated and coverslipped. Nuclear p53 immunostaining was assessed by image cytometry, using the Cytometrica program (C&V, Bologna, Italy). Staining was expressed as the percentage of labeled nuclear area over the total nuclear area of epithelial cells in the section labeling index according to Faccioli et al.³⁴

Immunohistochemistry was performed according to a conventional protocol (13 (link)). The paraffin-embedded prostate tissue sections were incubated in a dry oven at 63°C for 1 h. De-paraffinization and rehydration were then performed. The sections were incubated with 30–50 µl anti-IL-6 (SAB3300071), anti-TGF-β (SAB4502958) and anti-PCNA (WH000511M2) antibodies (dilution 1:100; Sigma-Aldrich; Merck KGaA) overnight at 4°C. The sections were washed with PBS buffer three times, each time for 5 min. A total of 30–50 µl HRP conjugated IL-6 (A0192), TGF-β (A0277) and PCNA (AF1363) secondary antibodies (1:1,000; Beyotime, Shanghai China) was added to the tissue section and incubated at 37°C for 1 h. The sections were washed with PBS buffer three times, each time for 5 min. Excess liquid was dried around the tissue and then placed flat into the moist chamber. A working solution of 3,3′-diaminobenzidene (30–50 µl; Sigma-Aldrich; Merck KGaA) was added to the sections and incubated at room temperature for 1–5 min. After the color was developed, the sections were rinsed with distilled water to stop the reaction. A positive results was defined as the presence of staining in 10% or more of cells. The stained tissue sections were reviewed and scored separately by two pathologists blinded to the clinical parameters.

Cytokine enzyme-linked immunosorbent assay (ELISA) kit (Genzyme, Cambridge, MA, USA), recombinant human IL-6 at a final concentration of 50 ng/ml (Sigma-Aldrich, Milan, Italy), proteasome inhibitor MG-132 at a final concentration of 10 mM (Calbiochem; Merck Chemicals Ltd., Nottingham, UK), cycloheximide at a final concentration of 20 µg/ml (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), hydroxyurea (Sigma-Aldrich; Merck KGaA), 5-FU (Fluorouracil; Teva Pharma Italia, Milan, Italy), Nutlin-3 (Sigma-Aldrich; Merck KGaA), anti-E-cadherin (clone 32A8, Cell Signaling Technology, Inc., Danvers, MA, USA), anti-IL-6 (Sigma-Aldrich; Merck KGaA), anti-p53 (Novocastra, Laboratories, Ltd., Newcastle upon Tyne, UK), and p53 Taqman gene expression quantitative assay kit (Applied Biosystems, Foster City, CA, USA) were used in the present study.