Immunohistochemical Analysis of Chorionic Villi

For immunohistochemistry assay, chorionic villi were submitted to the fixation step (in formalin 10%) and the dehydration step (in increasing alcohol concentrations) and embedded in paraffin, in parallel of the morphological analysis. Then, sections with 4μm were confectioned in microtome, placed on glass slides and subjected to immunohistochemical analysis (Gomes et al., 2011 (link); Castro-Filice et al., 2014 (link)). Briefly, sections were covered with citric acid pH 6.0 for 5min in a microwave for antigenic retrieval. The sections were incubated with 5% acetic acid solution for 8min at room temperature to block endogenous phosphatase activity and reduce the nonspecific binding. After, the sections were incubated with 2.5% goat serum for 45min at 37°C. The sections were incubated overnight at 4°C with Calomys callosus serum infected with T. gondii (1:100). On the following day, biotinylated goat-anti mouse IgG (1:600, Jackson Immuno Research Laboratories, West Grove, PA, United States) secondary antibody was added to the section for 1h 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; Gomes et al., 2011 (link); Castro-Filice et al., 2014 (link)).