Parasites were cultured in human foreskin fibroblasts seeded on a glass coverslip. For mitotracker treatment, the cells were treated with a 50-nm mitotracker for 30 min at 37°C followed by 4x DMEM and 1x PBS wash prior to fixation. At a specified time point, the cells were fixed with 4% PFA for 20 min at RT, blocked, and permeabilized in PBS/0.2% Triton-X100/2% bovine serum albumin (blocking buffer) for 20 min, RT followed by incubation with primary antibodies for 1 h at RT: myc (1:1,000; Cell Signalling), HA (1:1,000; Sigma-Aldrich), TgTom40 (1:1,000; gift from Giel van Dooren, Australian National University, Canberra, Australia), TgMys ([Ovciarikova et al., 2017], 1:1,000), TgCPN60 ([Agrawal et al., 2009], 1:1,000), IMC1 (1:1,000; gift from Gary Ward, University of Vermont, Burlington, VT), TgMic5 (1:1,000; gift from David Smith, Moredun Institute, Penicuik, UK), TgRop7 (1:1,000; gift from David Smith), Ty (1:800; gift from Giel van Dooren), Ty (1:1,000; Thermo Fisher Scientific), TgENO2 ([Courjol et al., 2017], 1:1,000), and CDPK1 (1:10,000; gift from Clare Harding, University of Glasgow, Glasgow, UK). Cells were washed three times with PBS/TritonX-100 and then incubated with secondary antibodies for 45 min at RT in the dark: AlexaFluor 594 or 488 (1:500; Invitrogen). Cells were washed three times with PBS/TritonX-100 and mounted on glass slides with DAPI Fluoromount-G (Southern Biotech).

For staining of extracellular parasites, glass coverslips were covered by poly-L-lysine solution (Sigma-Aldrich) for a minimum of 1 min at RT and then allowed to air-dry. Naturally lysed parasites were put onto the coverslips and spun gently (100–300 × g for 1–3 min) to allow the parasites to adhere to the coated coverslips. The cells were fixed and stained as described above.