Prior to LVAD implantation, all patients received intravenous
inotropics because of hemodynamic deterioration. Cardiac medication was discontinued initially in all patients after LVAD implantation (except for aspirin), but resumed if necessary ( Table 1). Informed consent to participate in this study was obtained from all patients before LVAD implantation. The pre-LVAD biopsy (LV apical core) was obtained at the time of LVAD implantation. These biopsies were compared with LV tissue specimens of the explanted heart after HTx (post-LVAD), taken from the apical half buy PF-02341066 of the LV. All biopsies were directly frozen. Normal myocardial tissue was obtained from vital organ donors from which the heart could not be used because of noncardiac reasons (n=2) and from autopsy on patients with no pathology of the heart (n=3). These biopsies served as a control. For the immunohistochemistry (IHC) of integrins, only (monoclonal) antibodies were selected that showed a strong staining without aspecific background on myocardial tissues. Therefore, only a limited number of integrins could be tested by IHC. Three-step immunoperoxidase staining to detect the localization of various integrins (and perlecan) was performed on sections prepared from frozen heart tissue
samples obtained pre- and post-LVAD. Eight-micrometer-thick sections were mounted on silan-coated glass slides. Frozen sections were air dried at room temperature, fixed in acetone (10 min), washed in PBS/Tween-20 for 10 min, and incubated with the primary antibodies ( goat anti-integrin α-5; -anti-integrin α-6, and -anti-integrin α-7, mouse anti-integrin GSK2118436 molecular weight β-1D or rabbit anti-integrin β-6; Table 2) for 1 h at room temperature. Next, sections were washed in PBS/Tween-20 (10 min) and fixed in formalin (4%) to cross
link the antibody to the tissue. Endogenous peroxidase was blocked by incubation in a blocking buffer (20 min) followed by washing in PBS/Tween-20 (30 min), and the sections were incubated with appropriate PO-labeled secondary antibodies for 30 min at room temperature. All secondary antibodies had been absorbed before use with 10% normal human serum to avoid cross reaction to human IgG. After another washing step in PBS/Tween-20 (30 min), the sections were incubated with Rabbit HRP Phosphatidylinositol diacylglycerol-lyase Powervision (Immunologic, KliniPath, The Netherlands) for 30 min at room temperature. Finally, the slides were washed again in PBS/Tween-20 for 30 min and incubated in a 3.3.di-aminobenzidineterachloric acid (DAB) solution for 10 min (room temperature), washed with aqua dest (10 min), and counterstained with Mayer’s hematoxylin. Slides were dehydrated and mounted in Pertex. The intensity of the IHC staining was scored (in a blinded fashion by two observers using a grid created with Image J software for Windows) on a semiquantitative scale ranging from negative (score=0), till intermittent/mild staining (score=1), moderate/diffuse staining (score=3), and strong/continuous staining (score=5).