In order to detect intracellular T. gondii replication, we standardized a cell culture based immunocytochemical assay using IgY anti-STAg as primary antibodies. Briefly, HeLa cells (ATCC No. CCL-2) cultured in 24-well plates were incubated with tachyzoites of T. gondii RH strain in a multiplicity of infection (MOI) equal to 1, in RPMI-1640 medium supplemented with 10% fetal calf serum for 24 h at 37uC and 5% CO 2 atmosphere. After incubation, cells were fixed with 4% formaldehyde and permeabilized with 0.1% Triton-X 100, followed by the incubation with the specific IgY antibody (30 mg/mL) for 2 h at 37uC. The next step consisted of the incubation with rabbit anti-IgY antibody labelled with FITC (Sigma), diluted 1:600 in PBS plus 49,6diamidino-2-phenylindole (DAPI, Sigma) for 1 h at 37uC. The reaction was read in an inverted fluorescence microscope system (EVOS, AMG Microscopy Group, USA).