**3.2 Peroxidase**

Experimental assays using HRP as a tracer of fluid phase endocytosis in tachyzoites were performed for transmission electron microscopy analysis only. No labeling was observed in the parasites after incubation at 4°C for 20–30 min (data not shown). The endocytic capacity of tachyzoites was tested during incubation for periods ranging from 5 min to 2 h at 37°C. During the course of the experiments, localization of HRP-Au on the surface of the parasites was rarely observed showing a low association of tracer. The labelling profile was altered when we increased the concentration

#### **Figure 4.**

*Ultrastructure of tachyzoites incubated for 5 min to 2 h at 37°C with HRP-Au. Longitudinal sections of parasites revealed particles of the HRP-Au complex (arrowhead) inside rhoptries (R). It was common to observed HRP-Au particles in more than one rhoptries (R) in the same tachyzoite.*

of HRP-Au, revealing a greater association of colloidal gold particles on the surface of the tachyzoites (not shown). In all experimental assays performed at 37°C, the intracellular localization of HRP-Au in tachyzoites was exclusively into rhoptries (**Figure 4A**–**C**). Presence of HRP-Au particles was commonly observed in more than one rhoptry in the same parasite (**Figure 4A**–**C**).

#### **Figure 5.**

*Analysis by flow cytometry of the incubation of tachyzoites with Dextran-TRITC. A: Tachyzoites incubated with PBS. Graphic show the morphology of the parasites, in terms of size and granularity (FSC x SSC) and the region of analysis R1. (B) Negative control. (C-F) The kinetics of Dextran-TRITC incorporation showed a low percentage of labelled parasites over time*
