Immunohistochemical Evaluation of Tissue Parasitism

The tissue parasitism was evaluated in the organs by immunohistochemistry as previously described [28 (link)]. Deparaffinized sections were incubated at room temperature with phosphate buffered saline plus 3% non-fat milk (Nestlé, São Paulo, Brazil) to reduce nonspecific binding, and then incubated at 4 °C overnight with polyclonal anti-T. gondii serum obtained from Swiss mice infected with ME-49 strain diluted in 0.01% saponin. After incubation with biotinylated goat anti-mouse antibody (Sigma Chemical Co., St. Louis, MO, USA), the assay sensitivity was improved by avidin–biotin–peroxidase complex (ABC kit, PK-4000; Vector Laboratories, Inc., Burlingame, CA, USA). The reaction was developed with 0.03% H₂O₂ plus 3,3′-diaminobenzidine tetrahydrochloride (DAB; Sigma) for 5 min. The sections were counterstained with Harris haematoxylin and examined under light microscope using a 40× objective. The tissue parasitism was scored by counting the number of cyst-like structures and parasitophorous vacuoles from two hundred microscopic fields in the small intestine, and in 40 microscopic fields in the lung or liver tissue section.