Vector vip substrate kit for peroxidase

Neutrophil, monocyte and eosinophil activation is known to catalyse the formation of hypochlorous acid that reacts with proteins and induces tyrosine halogenation such as 3,5-dibromotyrosine (Di-BrY). Leukocyte-derived oxidative stress was assessed after 24h of reperfusion in frozen sections of the ischemic hearts by immunostaining for 3,5-dibromotyrosine using a mouse anti-dibromotyrosine monoclonal antibody (at 10μg/ml, AMS biotechnology, LTD, Abingdon, UK). To avoid any potential cross-reactivity with mouse heart antigens and to increase the specificity of the primary antibodies, the VECTOR M.O.M immunodetection kit and the VECTOR VIP substrate kit for peroxidase (Vector Laboratories, Inc. Burlingame, CA) were used, following the manufacturer’s instructions.

The immunohistochemistry was performed on 4 µm sections of formalin-fixed, paraffin-embedded tissues. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 30 min at room temperature. Sections were exposed to high-temperature antigen unmasking by incubation at 98 °C with citric acid buffer, pH 6.0. Tissue sections were incubated for 1 h at room temperature with cyclooxygenase 2 antibody, diluted 1:100 (COX-2—rats polyclonal antibody, ab15191, Abcam, Cambridge, UK), and revealed with the Vector VIP Substratekit for peroxidase (Vector Laboratories, Burlingame, CA, USA). To objectively evaluate the tissue reaction and expression of COX-2, the scoring scales presented in Table 4 were applied [9 (link)].