Powervision poly hrp ihc detection system protocol

RPN13 was stained by following PowerVision Poly-HRP IHC Detection system protocol (Leica Biosystem). FFPE sections were deparaffinized in xylene, followed by rehydration in graded ethanol. Antigen retrieval was performed by steaming specimens at 100 °C for 20 min in Target Retrieval Solution (Dako) and subsequently washed in Tris-buffered saline with Tween 20 (TBST, 0.05% Tween 20). Endogenous peroxidase was blocked, by treatment of slides with Dual Endogenous Enzyme-Blocking Reagent (Dako) for 5 min at room temperature. Sections were covered with 1:500 dilution of mouse monoclonal ADRM1 Antibody (F-12) supplied by Santa Cruz Biotechnology (sc-166,754) diluted with Antibody Dilution Buffer (ChemMate) and then incubated at room temperature for 45 min. Slides were then washed with TBST, followed by incubation with PowerVision Poly-HRP Anti-Mouse IgG for 30 min at room temperature. After three washes in TBST, sections were treated with DAB chromogen (3, 3 ‘-diaminobenzidine tetrahydrochloride; Sigma) for 20 min in the dark. Sections were counterstained with Mayer’s hematoxylin (Dako), dehydrated with ethanol and xylene, and mounted permanently.

CD3 was stained by following PowerVision Poly-HRP IHC Detection system protocol (Leica Biosystem). Briefly, FFPE sections were deparaffinized in xylene, followed by dehydration in graded ethanol. Antigen retrieval was performed by steaming specimens at 100°C for 20 min in Target Retrieval Solution (Dako) and subsequently washed in Tris-buffered saline with Tween 20 (TBST, 0.05% Tween 20). Endogenous peroxidase was blocked, by treatment of slides with Dual Endogenous Enzyme-Blocking Reagent (Dako) for 5 min at room temperature. Sections were covered with rabbit monoclonal CD3 primary antibody (ThermoFisher, RM-9107, 1:300) diluted with Antibody Dilution Buffer (ChemMate) and then incubated at room temperature for 45 min. Slides were then washed with TBST, followed by incubation with PowerVision Poly-HRP Anti-Rabbit IgG for 30 min at room temperature. After three washes in TBST, sections were treated with DAB chromogen (3, 3 '-diaminobenzidine tetrahydrochloride; Sigma) for 20 min in the dark. Sections were counterstained with Mayer’s hematoxylin (Dako), dehydrated with ethanol and xylene, and mounted permanently.