Anti α actin antibody

Briefly, flash-frozen aortic segments were homogenized by a glass homogenizer in protein extraction buffer [1% SDS, 10 mM EDTA, and complete mini protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland)]. Samples were heated to 85°C for 15 min and centrifuged at 15,000 ×g for 15 min at 4°C. Protein concentration of the supernatants was determined by use of the DC Protein Assay (Bio-Rad Laboratories, Hercules, CA). Samples were diluted with 6x Laemmli buffer (30% glycerol, 50 mM EDTA, 0.25% bromphenol blue, and 10%  β-mercaptoethanol) and heated to 85°C before loading. The protein samples (40 µg) were run on a 7.5% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and the separated proteins were transferred to polyvinyl difluoride membranes. Nonspecific-binding sites were blocked with 5% BSA. The membranes were then incubated with the monoclonal antibody to eNOS (1 : 1000) (BD Transduction Laboratories). Afterwards, membranes were incubated with anti-rabbit or anti-mouse immunoglobulin (IgG) peroxidase-conjugated secondary antibodies (1 : 2500). Bound antibodies were detected by ECL Western blotting detection kit (Amersham Life Sciences, Arlington Heights, IL, USA). To ensure equal protein loading, membranes were stripped and reprobed with anti-α-actin antibody (1 : 5000; Abcam).

Primary cultures of mouse enteric neurons submitted to the above-described conditions and grown on coverslips were fixed with 4% buffered paraformaldehyde at room temperature for at least 1 h. To identify neuronal bodies and neurites and facilitate their quantification we used anti-PGP 9.5 (Rabbit, 1:500; Cedarlane, USA), anti-β-tubulin III (Mouse, 1:800; Millipore, USA) and anti-nNOS (Rabbit, 1:500; Epitomics, USA). Smooth muscle cells were identified using the anti-α actin antibody (Rabbit, 1: 400; Abcam, USA). For the immune-labeling of other structures we used the following antibodies: anti-3-NO₂-tyrosine (Rabbit, 1:200; provided by Dr. Rafael Radi), an antibody that specifically detects the protein bound 3-Nitrotyrosine (NO₂Tyr) (Brito et al., 1999 (link)); anti-MnSOD2 (Mouse, 1:200; Santa Cruz, USA) and anti-T. cruzi (Rabbit, 1: 5,000; provided by Prof. Maria T. Bahia, UFOP). Identification of cell nuclei in vitro was performed by the fluorescence-emitting probe Hoechst H33342 (Invitrogen, USA), which binds to nuclear DNA and allows the optimal visualization of parasite and host nuclear morphology. The coverslips were analyzed under the Olympus BX51 fluorescence microscope and images were obtained using Image-Pro Express 4.0 software (Media Cybernetics, USA).