Anti α mhc

The myocardial expression levels of α- and β-myosin heavy chain (MHC), Bax, as well as Bcl-2 were detected using immunohistochemistry techniques. Sections (10 μm) from paraffin-embedded tissues were stained using the simplified histostain SP-kit (Zymed Laboratories, USA) according to the manufacturer's instructions. After deparaffinization and rehydration and the inhibition of endogenous peroxidase, the sections were exposed to primary antibodies at 4°C overnight. After incubation with a secondary antibody at room temperature, the sections were incubated in 3,3 N-diaminobenzidine tetrahydrochloride (DAB) and counterstained with hematoxylin. The following primary antibodies were used at a dilution of 1:150: anti-α-MHC, anti-β-MHC, anti-Bax and anti-Bcl-2 (Santa Cruz Biotechnology, CA, USA). The secondary antibody was goat-anti-mouse immunoglobulin conjugated with horseradish peroxidase (dilution of 1:200). Digital images of the stained sections were acquired using a Nikon ECLIPSE E100 microscope with a digital camera system. Unanimity regarding positive immunohistochemical staining in each preparation was reached by two blinded investigators using Image-Pro Plus 5.1 software (Media Cybernetics, Silver Spring, MD).