Pl fluotar 100 objective

1 × 10⁵ gametocytes at stages II, III, IV and V were loaded with 100 nM of the acidotropic fluorescent dye LysoTracker Red DND-99 in complete medium and incubated for 2 h at 37°C. During the last 15 min 10 μg ml⁻¹ of Hoechst 33258 was added to stain nuclei, followed by two washes in 1×PBS. A Leica DMRB microscope was used to visualize live samples. Fluorescence images were acquired using a Leica DFC340 FX camera through a Leica PL FLUOTAR 100× objective. Filters used to detect LysoTracker Red DND-99 were: EX: 515–560, EM: 590 long-pass filter. Filters used to detect Hoechst 33258 were: EX: 340–380, EM 425 long-pass filter.

3D7/pfULG8-GFP stage V gametocytes were fixed on a glass smear
with paraformaldehyde 4% – glutaraldehyde 0.075% for 10 minutes
and permeabilized with 0.1% Triton X-100 (Tonkin
et al., 2004). After 30 min preincubation in 1% BSA,
parasites were incubated for 1h with an anti-377B rabbit antiserum (1:800) (Alano et al., 1995 (link)). After incubation and washes
in 1×PBS, parasites were incubated with affinity purified, rhodamine-conjugated
secondary antibody against rabbit IgGs (1:800) and 1μg/ml Hoechst 33258. After
final washes, samples were observed in a Leica DFC340 FX camera through a Leica PL FLUOTAR
100× objective in a microscope equipped with filters BP 340–380 (Hoechst),
BP 515–560 (rhodamine) and BP 470–490 (fluorescein).