For histology, 4-μm sections of paraffin-embedded samples were stained with H&E. IL-27 production was assessed in formalin-fixed, paraffin-embedded tracheobronchial, pelvic, and mesenteric lymph nodes from autopsies of men aged 59–65 y whose death was not due to cancer, autoimmune, or inflammatory disease. Single and double immunohistochemistry was performed with the following Abs: anti–IL-27 (rabbit polyclonal LS-B2719; LifeSpan BioSciences, Seattle, WA), anti–IL-27Rα/WSX-1 (rabbit polyclonal; Novus Biologicals, Cambridge, U.K.), anti-CD68 (clone PG-MI; Dako, Glostrup, Denmark), anti-CD83 (clone IH4b; Novocastra, Newcastle, U.K.), and anti-CD11c (rabbit monoclonal EP1347Y; Abcam, Cambridge, U.K.).
For IL-27/CD68, IL-27/CD11c, IL-27R/CD11c, and IL-27/CD83 double stainings, paraffin-embedded tissue sections were deparaffinized, treated with H2O2 (3%) for 5 min to inhibit endogenous peroxidase, and then washed in H2O. They were then incubated for 30 min with rabbit anti–IL-27 Ab, followed by detection with a Bond polymer refine detection kit (Leica Biosystems) according to the manufacturer’s protocol, and subsequently incubated for 30 min with the second primary Ab (mouse anti-CD68, rabbit anti-CD11c, and mouse anti-CD83) followed by detection with a Bond polymer refine red detection kit (Leica Biosystems), according to the manufacturer’s protocol.
For IL-27/CD68, IL-27/CD11c, IL-27R/CD11c, and IL-27/CD83 double stainings, paraffin-embedded tissue sections were deparaffinized, treated with H2O2 (3%) for 5 min to inhibit endogenous peroxidase, and then washed in H2O. They were then incubated for 30 min with rabbit anti–IL-27 Ab, followed by detection with a Bond polymer refine detection kit (Leica Biosystems) according to the manufacturer’s protocol, and subsequently incubated for 30 min with the second primary Ab (mouse anti-CD68, rabbit anti-CD11c, and mouse anti-CD83) followed by detection with a Bond polymer refine red detection kit (Leica Biosystems), according to the manufacturer’s protocol.