After the injection of the S. plumieri venom, the peak values of
MAP and HR were measured. Cross-neutralisation experiments were performed in order to determine buy EX 527 if stonefish antivenom (SFAV, obtained from CSL, Melbourne, Australia) was able to neutralise the nociceptive, edematogenic and cardiovascular effects induced by SpV. For neutralisation of nociceptive and edematogenic activities, samples of SpV were incubated at 25 °C for 30 min with SFAV at different ratios (1:0.25, 1:0.5, 1:1.0 and 1:1.5 μg of SpV/U of SFAV). After that, 30 μl of each mixture containing 15 μg of SpV were injected in the right hind paw of mice. Nociceptive and edematogenic activities were evaluated after 0.5 h according to items 2.2.1 and 2.2.2 (N = 4). For the cardiovascular assays, S. plumieri venom was pre incubated with SFAV (1:1 SpV/U of SFAV for 5 min at 25 °C), and subsequently, the mixture was administrated in bolus (300 μg protein of SpV/kg) according to
item 2.2.3 (N = 7). Samples of S. plumieri venom (15 μg and 300 μg), in appropriate vehicle, were submitted to the same incubation conditions (25 °C for 30 or 5 min) and used as positive control for inflammatory and cardiovascular assays, respectively. SpV (100 μg of protein) was applied to each of 7 cm immobilized linear pH gradients (pH 3–10 and Belinostat 4–7) strips (IPG, Bio-Rad), with Deastreak rehidration solution (Amersham, Uppsala, Sweden) for 12 h, 50 V at 20 °C. Isoelectric focusing (IEF) was performed in an IEFCell system (Bio-Rad, Hercules, CA). Electrical conditions were set as described
by the supplier. After the first-dimension run, the IPG gel strip was incubated at room temperature for 15 min in equilibration buffer (50 mM Tris–HCl pH 8.8, 6M urea, 2% SDS, 30% glycerol and traces of bromophenol blue) containing 125 mM DTT, followed by a second incubation step (15 min at room temperature) in equilibration buffer containing 125 mM iodacetamide instead Demeclocycline of DTT. The second dimension electrophoresis was performed in a vertical system with uniform 10% separating gel (mini PROTEAN 3 cell; Bio-Rad) at 25 °C, according to the method described by Laemmli (1970). Protein spots in the gel were stained with colloidal coomassie blue brilliant CBB G-250 following procedures described elsewhere (Neuhoff et al., 1988). S. plumieri proteins separated by 2D electrophoresis (according to item 2.3) were transferred to a nitrocellulose membrane for 1 h at 350 mA/100 V. Membrane was blocked in 5% low fat milk, 0.3% tween 20 in phosphate buffered saline (PBS). Following blockade, membrane was washed with PBS and probed (1 h at 25° C) with a 1:500 dilution of stonefish antivenom. Another washing step was performed and the bound antibodies were probed (1 h, 25 °C) with a diluted peroxidase-conjugated antibody (1:5000 in PBS containing 0.05% tween 20).