Axioskop 2 epifluorescence microscope

Zombie Red (1:1,000) and 4.5 µg/ml of fluorescein isothiocyanate (FITC)-annexin V (BioLegend) in annexin V binding buffer (BioLegend) were used to stain parasites grown without serum at 0 h, 16 h, and 24 h, and the cells were subjected to FACS analysis using a BD LSRFortessa cell analyzer. The live parasites were gated by excluding positive Zombie Red signal. Apoptotic levels were determined by analysis of the mean fluorescence intensity (MFI) of annexin V of live parasites. Parasites treated with dimethyl sulfoxide (DMSO) were used as the healthy control, and parasites treated with 4 µM staurosporine (STS) for 16 h composed the apoptotic control.
DNA fragmentation was done by incubating EV and TvMIF-OE in serum-free Diamond’s media and fixing parasites in 4% formaldehyde–PBS. Parasite nuclei were stained with ProLong Gold antifade mountant with 4′,6-diamidino-2-pheylindole (DAPI) (Thermo Fisher Scientific). The slides were then imaged using an Axioskop 2 epifluorescence microscope (Zeiss). The percentage of fragmented DNA was determined by the number of fragmented DNA in a total of 300 nuclei counted in each sample by using ZEN lite software.

Brains were removed and quick-frozen in liquid nitrogen. Cryo-sectioning was used to produce 10 µm sagittal sections, which were placed on Superfrost Plus positively charged microscope slides. Brain sections were fixed for 5 min in ice-cold 4% (v/v) paraformaldehyde in phosphate-buffered saline (PBS). Sections were then permeabilized with 0.1% (v/v) Triton X-100 in PBS for 30 min at room temperature (RT). Tissue sections were blocked in 20% (v/v) normal goat serum in PBS for 30 min and incubated overnight at 4 °C with primary antibody (PHF1 and 12E8). The primary antibodies were diluted 1:200 in PBS containing 0.5% lambda-carrageenan (Sigma Aldrich, Munich, Germany, 22049) and 0.02% sodium azide and applied overnight to the sections at 4 °C. Following a washing step, brain sections were incubated with Cy3-conjugated anti-rabbit antibody diluted 1:300 in PBS with the same additions as above for 1 h at RT. Finally, antibody-labeled brain sections were embedded in Fluoromount G medium with DAPI (Electron Microscopy Sciences, Hatfield, PA, USA for microscopic analysis (Zeiss Axioskop 2 epi-fluorescence microscope equipped with a digital Zeiss AxioCamHRc camera, Carl Zeiss Jena, Jena, Germany).

Alkaline comet assays were carried out using the Trevigen CometAssay electrophoresis kit according to manufacturer’s instructions. Briefly, cells were embedded into low melting agarose on comet slides and incubated in lysis solution overnight in the dark at 4°C. They were incubated in a solution of 300 mM NaOH, 1 mM EDTA for 30 minutes at room temperature, then electrophoresed in this solution for 30 min at 21 volts at 4°C in the dark. Comet slides were immersed in 70% ethanol for 10 min at room temperature and dried at 37°C for 15 minutes. They were then stained with 1x SYBR gold in TE buffer (pH 7.5) for 30 minutes at room temperature, dried for an additional 15 minutes at 37°C and visualized with a Zeiss Axioskop 2 epifluorescence microscope with a 10x objective. Data were analysed with CaspLab 1.2.3 software.