Lsh 710 confocal microscope

NbAn33-chNPs (1 mL at 1 mg·mL^-1) were labelled using the Alexa Fluor 594 Protein labeling kit (Invitrogen), in 5 mM KCl, 80 mM NaCl, 1 mM MgSO₄, 20 mM Na₂HPO₄, 2 mM NaH₂PO₄, pH 7.4) at room temperature for 1 h. Labelled NP were washed three times by centrifugation at 14,000 g for 30 min and resuspended in 5 mM KCl, 80 mM NaCl, 1 mM MgSO₄, 20 mM Na₂HPO₄, 2 mM NaH₂PO₄, 20 mM glucose, pH 7.4, at a final concentration of 1 mg·mL^-1. Live bloodstream forms (10⁶ cells·mL^-1) were incubated in 5 mM KCl, 80 mM NaCl, 1 mM MgSO₄, 20 mM Na₂HPO₄, 2 mM NaH₂PO₄, 20 mM glucose, pH 7.4 (TDB) with Alexa 594 labelled NbAn33-chNPs (50 μg·mL^-1) for 10 min at 37°C. NPs excess was removed by centrifugation at 4°C. Parasites were resuspended in TDB with tomato lectin-FITC conjugate (Sigma) at 20 μg·mL^-1, incubated for either 2 or 10 min at 37°C and then fixed in 4% paraformaldehyde in PBS for 1 h at 4°C. Finally, trypanosomes were washed with PBS three times, spread on poly-L-lysine-coated slides, and mounted in DAPI-containing Vectashield medium (Vector Laboratories). For fluorescence microscopy analysis image acquisition was performed with a LSH 710 Confocal Microscope (Zeiss) and image analysis with ZEN 2012 (Zeiss) software.

Bloodstream forms of T. brucei treated with curvicollide D or dimethyl sulfoxide (DMSO) (control) were washed with PBS at pH 7.5. Parasites were fixed in 4% (w/v) paraformaldehyde (PFA) (Sigma-Aldrich, P6148) and permeabilized with a solution containing 0.2% triton X-100 (Sigma-Aldrich, T8787) in PBS buffer as previously describe [59 (link)]. For immunofluorescence, fixed cells were treated with the primary antibody anti L1C6 (mouse) (kindly provided by Keith Gull) in blocking buffer containing 3% bovine serum albumin (Sigma-Aldrich, A7906) during 1 h at room temperature. Samples were then incubated with the secondary antibody goat anti-mouse IgG Alexa 488 (Invitrogen, Waltham, MA, USA, A11001) in the same blocking buffer. On the other hand, a group of fixed cells treated with curvicollide D or DMSO were only coated in a poly-L-lysine-coated slides (Thermo Fisher Scientific, Waltham, MA, USA, J2800AMNZ) and mounted in DAPI-containing Vectashield (H-1200, Vector) (Vector laboratories, Burlingame, CA, USA). Image acquisition was performed with a confocal LSH 710 Confocal Microscope (Zeiss) and image analysis with ZEN blue (Zeiss) software.