Ep1551y

Frozen tissue blocks were cut at 5 µm thickness and picked up on X-tra^® Slides (Leica Biosystems). After air-drying for 30 min at room temperature, sections were immediately fixed with acetone at −20°C for 20 min. Slides were rehydrated in 1× PBS for 10 min. To quench endogenous peroxidase activity, sections were incubated with peroxidase reagent (3% H₂O₂ in 1× PBS) for 15 min and gently washed twice in 1× PBS for 5 min. Slides incubated with a primary antibody cocktail overnight at 2°C to 8°C. The cocktail contained both monoclonal mouse anti-human CD3, (Clone F7.2.38; 1:100, Dako) and anti-p16 ARC antibody (EP1551Y; 1:100, Abcam, ab51243). Upon finishing and rinsing, the secondary antibody cocktail [peroxidase anti-rabbit IgG (H+L) (Vector Laboratories, PI-1000) and alkaline phosphatase anti-mouse IgG (H+L) (Vector Laboratories, AP-2000)] was prepared and applied as recommended by manufacturer (Vector Laboratories) followed by the subsequent visualization of AP activity (Vector Red Alkaline Phosphatase Substrate Kit) (Vector Laboratories, SK-5100) and HRP activity (Vector DAB Peroxidase Substrate Kit) (Vector Laboratories, SK-4100). Stained tissues were mounted with hematoxylin (Sigma-Aldrich), dehydrated, covered and imaged via microscope.