Effects of harmane, norharmane and harmine on parasite invasion and replication. A. Extracellular tachyzoites (2.5×106) were treated for 1 h with different doses of the βCs, washed and allowed to invade Vero cells monolayers for 1 h at 37°C, and then fixed and incubated with murine anti-SAG1 antibody. The secondary antibody used was Alexa Fluor 594 goat anti-rabbit. For each treatment, the number of parasitophorous vacuoles (PVs) per field was counted. Fifty fields selected at random were analyzed in duplicate. B. Infected monolayers were treated for 24 h with different doses of the βCs, fixed and immunostained with anti-SAG1 antibody. The number of tachyzoites inside the PV was counted. Fifty fields selected at random were counted in duplicate. In total, approximately 1000 PVs per dose were analyzed in A and B. These panels are representative of three independent experiments. All the experiments presented similar results.