Journal: PLoS ONE
Article Title: Cardiac-Oxidized Antigens Are Targets of Immune Recognition by Antibodies and Potential Molecular Determinants in Chagas Disease Pathogenesis
Figure Lengend Snippet: Oxidative adducts are enhanced in experimental and human myocardium during Chagas disease. ( A ) Sprague-Dawley rats (or C3H/HeN mice) were infected with T. cruzi, and cells were harvested at day 40 (acute stage) and 180 (chronic stage) post-infection. Heart homogenates were resolved on 10% acrylamide gels, and Western blotting was performed with specific antibodies to detect 4 hydroxynonenal (4-HNE, panel a ), malondialdehyde (MDA, panel b ), dinitrophenyl (DNP)-derivatized carbonyl ( panel c ), and 3-nitrotyrosine (3NT, panel d ) adducts. Coomassie blue staining of membranes ( panel e ) confirmed the equal loading of samples. ( B ) Cryostat sections of human cardiac biopsies (5-µm) from normal healthy donors ( panels a, c, e ) and chagasic patients ( panels b, d, f ) were submitted to immunohistochemistry as described in Materials and Methods . Shown are representative images of immunostaining with anti-4-HNE antibodies ( panels a, b ). Tissue sections were incubated with DNPH to derivatize carbonyl proteins, and immunostaining was performed with anti-DNP antibody ( panels c–f ). HRP-conjugated ( panels a–d ) and rhodamine-conjugated ( panels e, f ) secondary antibodies were utilized to capture the color (brown) or fluorescence signal, respectively.
Article Snippet: Membranes were probed with sera from normal or chagasic rats, mice or human patients (1∶100 dilution) followed by HRP-conjugated secondary antibody (1∶5000, BioRad), and signal was detected by an ECL plus chemiluminiscence detection system (GE-Healthcare).
Techniques: Mouse Assay, Infection, Western Blot, Multiple Displacement Amplification, Staining, Immunohistochemistry, Immunostaining, Incubation, Fluorescence