Il 1β

Paraffin embedded myocardial samples were dewaxed and rehydrated in xylene and ethyl alcohol followed by incubation in 0.3% methanol/H₂O₂ to block endogenous peroxidases. After antigen retrieval was performed in citrate buffer (pH 6.0, 95–100 °C), the sections were incubated overnight with the following primary antibodies (α-SMA,CD31, 1:200, Cell Signaling Technology, USA; TGF-β, TNF-α, IL-1β, Abcam, USA) at 4 °C. Two-step technique (SuperPictureTM3rd Gen IHC Detection kit; Invitrogen, CA, USA) was used for visualization, using 3, 3′-diaminobenzidine (DAB, 0.1 mg/ml, 0.02% H₂O₂, Vector Laboratories, Burlingame, USA) as the chromogen. Sections were counterstained with hematoxylin. We selected the same slide which contained the largest infarct size for each group to ensure the comparability of the three groups. For each slide, 5 separate fields were examined randomly and digitized by microscopy at a magnification of 400×. All five fields were in the same position in all three groups. Image-Pro Plus software (Media Cybernetics, Rockville, USA) was used to determine the area of α-SMA, CD31, TGF-β, TNF-α and IL-1β positive staining.