Hrp conjugated anti digoxigenin antibody

Terminal Transferase dUTP Nick End Labeling (TUNEL) Assay was performed using the Apotag kit from Millipore with minor modifications. Briefly, five micron sections from formalin fixed paraffin embedded tissues were de-paraffinized with xylenes and rehydrated through a graded alcohol series. HIER was performed by immersing the tissue sections at 98 °C for 20 min in 10 mM citrate buffer (pH 6.0) with 0.05% Tween. Slides were pretreated with 3% hydrogen peroxide at room temperature, 10 mM Sodium Citrate at 65 °C, and Equilibration Buffer at RT. Slides were exposed to terminal transferase and digoxigenin labeled dUTP in Reaction Buffer for 1 h at 37 °C, stopped in Wash Buffer and blocked with 10% normal goat serum at RT. Slides were exposed to 1:2100 dilution of HRP-conjugated anti-digoxigenin antibody (Roche) for 1 h and DAB chromagen (Dako) for 5 min. Slides were counterstained with Hematoxylin (Fisher, Harris Modified Hematoxylin) at a 1:9 dilution for 2 min, blued in 1% ammonium hydroxide for 1 min, dehydrated, and mounted with Acrymount. For negative controls, consecutive sections were treated identically except that the terminal transferase enzyme was omitted and replaced by tris buffered saline in the reaction mixture. Images were captured using an Olympus DP70 camera on an Olympus BX61 microscope.