Fast red naphthol

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)).

Whole-mount in situ hybridizations (WMISHs) were performed according to Quiring et al. [83 (link)]. WMISHs for confocal imaging were stained with FastRed Naphthol (Sigma-Aldrich).

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)).

Whole-mount in situ hybridization (WMISH) was performed according to Quiring et al. [38 (link)] and Heermann et al. [18 (link)]. WMISHs for confocal imaging were stained with FastRed Naphthol (Sigma-Aldrich). The nuclei were stained with DAPI (4 μg ml⁻¹). For each in situ 8–10 embryos were stained, 4–6 were confocally imaged.