Dna dye hoechst 33342

To study liver-stage development, in vitro cultured human hepatoma (Huh7) cells34 (link) were infected with 10,000 sporozoites using standard techniques as described previously25 (link). After 24, 48 or 70 h, liver-stage parasites were fixed and permeabilized with ice-cold methanol and stained with the DNA-dye Hoechst 33342 (Invitrogen), rabbit anti-UIS4 antibodies (1:500 dilution35 (link)), and mouse anti-HSP70 antibodies (1:300 dilution36 (link)). Primary antibodies were detected by fluorescently labelled goat anti-mouse/rabbit IgG Alexa Fluor 488/546 conjugated antibodies (1:1,000 dilution, Invitrogen). Liver-stage parasites were counted and images recorded on a Zeiss AxioObserver Z1 epifluorescence microscope, equipped with a Zeiss AxioCam MRm camera, and processed minimally with Fiji31 (link).

Blood-stage development of selected mtp⁻ lines was analysed using a flow cytometry-based intravital competition assay, growing recombinant parasite lines in competition with a strongly fluorescent reference strain (Beryellow or Berred) within a single mouse30 (link). Data from the exponential growth phase, that is, with parasitaemia <1%, fitted a linear regression well (r²≥0.99) and allowed the calculation of the PMR from the slopes. For live protein localization, a drop of tail blood from an infected mouse was mixed with 200 μl pre-warmed RPMI 1640 complemented with 0.2 μl of the DNA-dye Hoechst 33342 (Invitrogen) and distributed onto poly-L-lysine coated cover slips. Cells were allowed to settle for 5 min at 37 °C. Next, cover slips were washed three times with pre-warmed RPMI, inverted and sealed. Images were recorded on a Zeiss AxioObserver Z1 epifluorescence microscope, equipped with a Zeiss AxioCam MRm camera, and processed minimally with Fiji31 (link).

Immunofluorescent staining of isolated HNE cells was performed using previously described protocols [25 (link)]. Isolated cells from HNE cell cultures in 1X PBS were allowed to settle on 1% Alcian Blue (Sigma-Aldrich Co, St. Louis, MO, Il, USA)-treated slides/cover slips for 20 min. on ice. Cells were fixed with 2% paraformaldehyde for 10 min. and washed with 1X PBS three times. Cells were permeabilized with 0.2% Triton X-100 in PBS for 15 min. Cells were incubated in blocking solution (5% donkey serum, 3% BSA, 0.1% Triton X-100 in 1X PBS) for 1 h at RT., followed by incubation in primary antibody diluted in blocking solution either for 1 h at RT or overnight at 4 °C. After washing with 1X PBS three times, cells were incubated in secondary antibody solutions for 2 h at RT. After washing with 1X PBS four times, slides were mounted with ProLong Diamond Antifade Reagent (Thermo Fisher, Waltham, MA, USA). Nuclei were stained using DNA dye Hoechst33342 (Invitrogen, Waltham, MA, USA). Slides were imaged with a Zeiss 800 upright confocal microscope with a 63X/1.4 oil objective. No detectable staining was observed for isotype-matched antibody or no primary antibody control slides. Using FIJI software (version 2.1.1), all images were processed, pseudo-colored, and brightness/contrast was adjusted identically across subjects.