Immunolocalization of Parasites in Villous Explants

To verify the immunolocalization of the parasites, villous explants were fixed in 10% buffered formalin, dehydrated in increasing alcohol concentrations, and embedded in paraffin. Sections with 4 μm were placed on glass slides and subjected to immunohistochemical analysis (de Oliveira Gomes et al., 2011 (link); Castro-Filice et al., 2014 (link)). Briefly, for antigenic retrieval, sections were covered with citric acid pH 6.0 for 5 min in a microwave. To block endogenous phosphatase activity and reduce the non-specific binding, the sections were incubated with 5% acetic acid solution for 8 min at room temperature and 2.5% goat serum for 45 min at 37°C, respectively. Next, sections were incubated overnight at 4°C with C. callosus serum previously infected with T. gondii (1:100). On the following day, biotinylated goat-anti mouse IgG (1:600, Jackson Immuno Research Laboratories, West Grove, PA) secondary antibody was added to the section for 1 h at 37°C. The reaction was developed with fast red naphthol (Sigma), the tissue counterstained with Harris's hematoxylin and analyzed under a light microscope (BX40, Olympus, Tokyo, Japan; de Oliveira Gomes et al., 2011 (link); Castro-Filice et al., 2014 (link)).