Figure 2. Schematic diagram representing the main steps of the application of the protocol for CL Brener and Y strains (epimastigote forms).

The halogenated thymidine analogs (CldU and IdU) are added separately in each culture for 12 h. After that, parasites must be washed with fresh media, mixed, and incubated for 24 h. Then samples must be washed, fixed, and added onto slides. Next, the parasite cells must have their DNA denatured and should be then processed for the detection of the thymidine analogs using primary antibodies (α-CldU and α-IdU) and the corresponding secondary antibodies (Alexa fluor 488 α-rat and Alexa fluor 568 α-mouse). Finally, mounting medium with DAPI must be added and the slides sealed.