After washing, the sections were incubated with biotinylated anti-mouse or anti-rabbit secondary antibody (Advanced™ HRP link, Dako®) for 30 min at room temperature, rinsed with PBS and Dasatinib incubated with Advanced™ HRP Enzyme for 30 min at room temperature. Horseradish peroxidase (HRP) activity was detected using the chromogenic substrate diaminobenzidine (DAB-Advanced™, Dako®). All incubations were
done in a humidified chamber. The antibody reactions were stopped by washing the slides with distilled water. The sections were counterstained with Ehrlich’s hematoxylin and then mounted in Canada balsam. For each antibody, a negative control was done by replacing the primary antibody with 1% PBS-BSA. Macrophages expressing
OPN was identified by double labeling the cells for CD68 and OPN. For this, the sections were incubated sequentially with both antibodies and then stained with the Envision Kit double stain system (Dako®). To quantify the immunoreactivity to OPN, two fields in the damaged area per section were evaluated (n = 6 animals per time-point, total of 12 fields/time-point). All field images were photographed with an Olympus BX51 photomicroscope using fixed parameters for light intensity, magnification (200×) and color (24 bits). The images were captured with a computer-aided image analysis system (Image Pro-Plus 4.0, Media Selleck TSA HDAC Cybernetics) connected to the photomicroscope. Image analysis (quantification of the optical density of immunoreactivity) was done using GIMP 2.6.4 software (GNU Image Manipulation Program, CNET Networks Inc.) that segmented the images by color. This segmentation by color made it possible to determine the percentage of pixels for staining by Methane monooxygenase a given antibody. CD68-positive macrophages were quantified by counting the number of positive cells
in ten fields in the damaged area per section (one section/rat and six rats/time interval, i.e., 60 fields per time interval) in the control and envenomed groups. Myogenin was quantified by counting the number of nuclei positive for the protein in muscle fibers. The immunohistochemical data were expressed as the mean ± S.D. Multiple comparisons were done using one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparisons test (GraphPad Prism 4.0 software, San Diego, CA, USA). Cell diameters (1 h post-venom vs. control group, regenerated fibers vs. intact fibers at 21 days post-venom and intact fibers in envenomed muscle vs. normal fibers in control PBS-injected muscle after 21 days) were compared using Student’s t-test. A value of p < 0.05 indicated significance.