Sp 5 mp confocal laser scanning microscope

Cells were washed in cold phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄-2H₂O and 1.5 mM KH₂PO₄), fixed for 30 min at 4 °C in 4% paraformaldehyde (PFA, Sigma-Aldrich, #47608) and washed in PBS. Cells were permeabilized in 0.5% Triton-X-100 (Plusone, #17-1315-01) for 10 min and blocked in 5% Bovine Serum Albumin (BSA, Sigma Aldrich #A7906) for 30 min. Primary antibodies were added over night at 4 °C and secondary antibodies for 1 h at room temperature. Cells were treated with DAPI (Invitrogen, Waltham, USA, #C10595) for 5 min to stain nuclei, washed in 1% BSA and mounted in 2% N-propyl-galleate (Sigma-Aldrich, #P-3130) in PBS/glycerine. Images were collected on an Olympus BX63 upright microscope with a 60 ×/1.35 NA oil objective using Olympus CellSens dimension software or (confocal images) on a Leica SP 5 × MP confocal laser scanning microscope with a 40 × or 60 ×/1.3 NA oil objective using Leica LAS AF software. Z-stacks were captured in intervals of 0.2 µm with corresponding Z-scans. Images were processed in ImageJ. No labeling was detectable in the absence of primary antibody at the settings used (data not shown).

Parasite proteins were extracted with 1% Triton in PBS supplemented with protease inhibitors. Protein extracts were boiled under reducing conditions in Laemmli buffer, separated using 10% SDS-PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane and detected using the rabbit α-PbP47 and a goat α-GFP (Rockland chemicals) antibodies at 1:100 and 1:1000 dilutions, respectively. Secondary horseradish peroxidase conjugated donkey α-goat IgG (Abcam) and goat α-rabbit IgG (Promega) antibodies were used at 1:5000 and 1:10000 dilutions, respectively. Gametocytes and ookinetes were fixed in 4% para-formaldehyde (PFA) in PBS for 20–25 min, smeared on glass slides and air-dried prior to blocking and antibody staining. Dissected midgut tissues were fixed after blood bolus removal in 4% PFA in PBS for 30 min, washed three times in PBS for 10 min, and blocked and permeabilized in 1% BSA and 0.2% Triton in PBS prior to antibody staining. Rabbit α-PbP47, rabbit α-TEP1^{29 (link)} and mouse α-P28 were used at 1:100, 1:300 and 1:1000 dilutions, respectively. Secondary Alexa Fluor 647 goat α-rabbit and 568 goat α-mouse IgG antibodies (Life technologies) were used at 1:1000 dilution. Images were acquired using a Leica SP5 MP confocal laser-scanning microscope, processed by deconvolution using the Huygens software and visualized with ImageJ.