Pcna af1363

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.