stopped by fixation with 4% paraformaldehyde. Labelling with antibodies against PbCSP (see below) preceded Triton X-100 permiabilisation [69] to identify sporozoites mid way through invasion, followed by re-fixation and permiabilisation for intra- cellular actin labelling. T. gondii tachyzoite invasion was captured using a potassium shift protocol (Kafsack et al., 2004). For JAS treatment of invasion, filtered P. falciparum and P. berghei merozoites were allowed to invade erythrocytes shaking at 37uC for 1.5 and 2 min respectively before treatment with 5 mM JAS then returned to invasion assay conditions for a further 1.5 or 2 min. Indirect immunofluorescence assay (IFA), electron microscopy and 3D SIM imaging Immunofluorescence microscopy (IFA). P. falciparum merozoites, P. berghei merozoites or sporozoites allowed to invade were fixed in solution and prepared for IFA as described [69] using 0.0075% glutaraldehyde/4% paraformaldehyde (ProSciTech, Australia) in PBS for merozoites, 4% paraformaldehyde in PBS for sporozoites, or (for ring stages) by cold methanol fixation [13]. For JAS treatment of rings P. falciparum late stage cultures were allowed to invade erythrocytes before commencing treatment with 1 mM JAS for 6 hours prior to imaging. Primary antisera in 3% BSA/PBS included rabbit anti-Act239–253 [1:300]; mouse anti-Act239–253 [1:100]; mouse anti-PfRON4 [70]; mouse anti-PbCSP [1:5000] [71]; mouse anti- Pb28 [72] [1:10,000]; rat anti-ERD2 [1:200] (MR4, ATCC Manassas Virginia); mouse anti-TgSAG1 (DG52) (Morisaki et al., 1995) [1:5,000] (a kind gift from L. D. Sibley, Washington University School of Medicine, USA); and mouse anti-Ty (Bastin et al., 1996) [1:1000]. Following washes appropriate secondary antibodies (Alexa Fluor-488, 594, Invitrogen) were at 1:500 before mounting in VectaShieldH (Vector Laboratories) with 0.1 ng/mL 49,6-diamidino- 2-phenylindole, DAPI (Invitrogen). Fluorescence images were obtained using a Plan-Apochromat 1006/1.40 oil immersion Phase contrast lens (Zeiss) on an AxioVert 200 M microscope (Zeiss) equipped with an AxioCam Mrm camera (Zeiss). Z-stacks were taken well above and below parasites and processed using the Axiovision release 4.7 or 4.8 deconvolution software package. Ultrastructural electron microscopy. P. berghei ookinetes (untreated or 1 mM JAS treated) were fixed in suspension with a freshly prepared solution of 2.5% glutaraldehyde in 0.1 M phosphate buffer (PB) (pH 7.4) for 1 hr on ice. These were washed (63) and fixed in 1% osmium tetroxide (ProSciTech, Australia) in 0.1 M phosphate buffer for 1 hr. Following extensive rinsing in distilled water samples were stained with 2% uranyl acetate (SPI-Chem, Australia) in water for 1 hr. Washed samples were dehydrated with ethanol and embedded in LR Gold Resin (ProSciTech,Australia). Following polymerisation by benzoyl peroxide (SPI-Chem, USA) ultrathin sections (80–90 nm) were cut on a Leica Ultracut R ultramicrotome (Wetzlar). After double contrasting with uranyl acetate and lead citrate sections were examined at 120 kV on a Philips CM120 BioTWIN Transmission Electron Microscope. Immunoelectron microscopy. Free or invading P. falciparum merozoites [41] or P. berghei ookines were fixed in 1% glutaraldehyde (ProSciTech, Australia) on ice for 30 min. Samples were pelleted in low-melt agarose before being transferred into water. Dehydration and sectioning was as above. For labelling, sections were blocked in PBS containing 0.8% (wt/vol) bovine serum albumin and 0.01% (wt/vol) Tween 80 and then incubated in anti-Act239–253 diluted in the above-mentioned solution. Samples were washed and incubated with secondary antibodies conjugated to 10 nm diameter gold particles (BioCell). Post- staining with 2% aqueous uranyl-acetate and 5% triple lead prior to imaging. Actin Filaments in Plasmodium Development 3D structured illumination microscopy (3D SIM). Samples were prepared as for IFA mounted in VectaShieldH (Vector Laboratories). Imaging was performed using a DeltaVision OMX 3D Structured Illumination Microscopy SystemH (OMX 3D-SIM, Applied Precision Inc, Issaquah, USA) as described [41]. Image processing and actin quantification Deconvolved Z-stacks were reconstructed in 3D, with interpo- lation, using Imaris version 7.1.0 (Bitplane Scientific). For clarity of display, gamma settings were altered on 3D reconstructions after deconvolution, however no comparisons of labelling levels were made from such altered images. Maximum and total actin fluorescence calculations were performed in Metamorph version 7.7.0 (Molecular Devices), within masked regions determined using the ‘‘auto threshold for light objects’’ function on the CSP labelling channel of sporozoites. Statistics were calculated using a student’s t-test in Prism version 5 (GraphPad). General image handling was undertaken using either Image J or Adobe Photo- shop CS4. Final images were assembled in Adobe Illustrator CS4 for figure generation. Supporting Information Figure S1 Serial section, transmission electron micrographs with anti-Act239–253 (rabbit) immunogold labelling (arrowheads) of free P. falciparum merozoite. Scale bar = 0.2 mm. (TIF) Figure S2 Transmission electron micrographs of untreated and 1 mM JAS treated P. berghei ookinetes. Scale bar = 0.5 mm. (TIF) Figure S3 A) Western blot of P. falciparum schizont lysate fractionated by hypotonic lysis with subsequent carbonate extraction: labelling with anti-MTRAP (a membrane bound control) and PfADF1 (a cytosolic control). P = pellet fraction; S = supernatant fraction. B) Widefield IFA of intracellular T. gondii tachyzoites within HFF cells labelled with rabbit anti-Act239–253. Scale bar = 5 mm. (TIF) Figure S4 3D reconstruction of widefield IFA with deconvolu- tion of independent sporozoites treated with 1 mM JAS and labelled with PbCSP (Red), rabbit anti-Act239–252 (Green) and DAPI (Blue). Grid = 1 mm. Asterisk marks parasite apex. See also Movie S1. Gamma settings were altered in 3D reconstructions. (TIF) Movie S1 Rotation of immunofluorescence imaging of 1 mM JAS treated P. berghei sporozoite, labelled with anti-PbCSP (Red), rabbit anti-Act239–253 (Green) and DAPI (Blue). See Figure 2. (MOV) Movie S2 Rotation of immunofluorescence imaging of 1 mM JAS treated P. berghei ookinete, labelled with Pbs28 (Red), rabbit anti-Act239–253 (Green) and DAPI (Blue). See Figure 2. (MOV) Movie S3 Rotation of 3D-SIM imaging of P. berghei ookinete, labelled with Pbs28 (Red), rabbit anti-Act239–253 (Green) and DAPI (Blue). See Figure 2. (MOV) Movie S4 Rotation of 3D-SIM imaging of P. berghei ookinete following 1 mM JAS treatment, labelled with Pbs28 (Red), rabbit anti-Act239–253 (Green) and DAPI (Blue). See Figure 2. (MOV) PLoS ONE | www.plosone.org 12 February 2012 | Volume 7 | Issue 2 | e32188