, also described that click here the inflammatory reaction induced by skin mucus was characterized by antigen persistence in the peritoneal cavity that allowed the activation of phagocytic cells with capacity of antigenic presentation. However, the compositional differences and biological functions of fish skin mucus and the sting venom from the catfish C. spixii have not

been investigated. Thus, the present study was conducted to gain a better understanding of the peptide and protein components of fish skin mucus and the sting venom from the catfish C. spixii. The biological functions of both types of components were investigated during microcirculation in mice using an intravital microscopy that allows the visualization of extremely rapid adhesion events at the interface between blood and tissue in living animals. Male Swiss mice (5–6 weeks old) were obtained from a colony at the Butantan Institute, São Paulo, Brazil. Animals

were housed in a laminar flow holding unit Selleck Forskolin (Gelman Sciences, Sydney, Australia) on autoclaved bedding, in autoclaved cages, in an air-conditioned room under a 12 h light/dark cycle. Irradiated food and acidified water were provided ad libitum. All procedures involving animals were in accordance with the guidelines provided by the Brazilian College of Animal Experimentation. C. spixii specimens were captured with a trawl net from the muddy bottom of Paranaguá Bay (Pontal do Sul, Paraná State, Brazil), and fish were anesthetized with 2-phenoxyethanol prior to sacrifice

( Tsutsui et al., 2005). Stings (dorsal and pectorals) were cut off at their bases with cutter pliers and immediately taken to the laboratory to prepare the pools of each venom. Florfenicol The skin mucus was obtained by scratching the skin with a glass slide, and was immediately conditioned in ice, and then diluted in sterile saline, homogenized, and centrifuged for collection of the supernatant. The sting venom extraction was accomplished with trituration and centrifugation. The supernatant was collected and stored at −70 °C ( Junqueira et al., 2007; Subramanian et al., 2007). Protein concentrations were determined by the colorimetric method of Bradford (1976) using bovine serum albumin (Sigma Chemical Co., St Louis, MO) as standard protein. Endotoxin content was evaluated (resulting in a total dose < 0.8 pg LPS) with QCL-1000 chromogenic Limulus amoebocyte lysate assay (Bio-Whittaker) according to the manufacturer’s instructions. Sting venom or skin mucus (100 μg of each sample) were reconstituted separately in ammonium bicarbonate buffer (100 mM, pH 8.5) and 3 μL of DTT (100 mM, Sigma–Aldrich, St. Louis, MO, USA). The mixture was incubated for 30 min at 37 °C. To alkylate the protein, 7 μL of iodoacetic acid (100 mM in 50 mM CH5NO3, Sigma–Aldrich, St. Louis, MO, USA) were added and the mixture was incubated for an additional 30 min at room temperature in the dark.