Lsm 510 meta confocal microscopy

Approximately 10⁶ promastigotes of LmjF and pTRP transfectants in the stationary growth phase were washed with PBS and adhered to cover slips treated with poly-L-lysine (Sigma-Aldrich, St. Louis, MO, USA) for 15 minutes. The cells were then fixed with 3% paraformaldehyde for 10 minutes and treated with 50 mM ammonium chloride for 10 minutes. The fixed cells were permeabilized and blocked with 0.1% Triton X-100 and 0.1% BSA in PBS for 10 minutes at room temperature. To analyze sub-cellular TRP localization, anti-TRP polyclonal antibody (1:100 dilution) was visualized using an anti-rabbit secondary antibody conjugated to Alexa488 (Life Technologies, Carlsbad, CA, USA) (1:500 dilution). Anti-BiP/GRP78 (BD Bioscience, Iowa, USA) (1:500 dilution) was visualized using an anti-mouse secondary antibody conjugated to Alexa594 (Life Technologies, Carlsbad, CA, USA) (1:500 dilution). Nuclear and kinetoplast DNA were labeled using DAPI. Each step was followed by washing with PBS 10 times. The coverslips were mounted in ProLong media (Life Technologies, Carlsbad, CA, USA). All imaging was performed at the Molecular Imaging Center (MIC) of the University of Bergen, using a Zeiss LSM 510 Meta confocal microscopy. Co-localization images were edited using Photoshop 6.

Fluorescently-stained raft-exhibiting giant liposomes were prepared essentially according to a previous report25 . The films of the lipid mixture (DOPC/DPPC/cholesterol or 1/_D-(+)-glucose = 2:2:1:15) with 0.2% Rhodamin-DHPE and 1% GM₁ was prepared in glass tubes, and hydrated with deionized water, followed with incubation at 37 °C for one hour to form giant vesicles. The liposome suspension was combined with a solution of fluorescein-labeled CtxB-488 for staining the cholesterol-rich raft-like domain. The fluorescent images of liposomes were acquired using a confocal fluorescent microscope (LSM 510 META confocal microscopy, Carl Zeiss, Jena, Germany).

Excess accumulation of lipid bodies within parasites was detected by using Nile Red dye^{55 (link)}. Briefly, promastigotes (1 × 10⁷ cells/ml) were treated with β-sitosterol_CCL (IC₅₀ dose) at 24 h and 48 h. Cells were incubated with 10 μg/ml Nile Red for 30 min in the dark and then fixed with 4% paraformaldehyde in phosphate buffer (0.1 M, pH 7.2). Parasites were then imaged under LSM510-META confocal microscopy (Carl Zeiss, Germany)^{56 (link)} at 480 nm for excitation and 530 nm for emission.