Indirect immunofluorescence assays (IFA) were performed on freshly collected tachyzoites washed with buffer A with glucose (BAG, 116 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO4, 50 mM HEPES, pH 7.2, and 5.5 mM glucose) and fixed with 4% formaldehyde for 1 h, permeabilized with 0.3% Triton X-100 for 20 min, and blocked with 3% bovine serum albumin (Miranda et al., 2010 (link)). Immunofluorescence was performed as previously described (Miranda et al., 2010 (link)) and primary and secondary antibodies concentrations are indicated in the figure legends. Fluorescence images were collected with an Olympus IX-71 inverted fluorescence microscope with a Photometrix CoolSnapHQ CCD camera driven by DeltaVision software (Applied Precision, Seattle, WA). Collected images were deconvolved using Softworx deconvolution software (Applied Precision, Seattle, WA). For all images, 15 cycles of enhanced ratio deconvolution were used.
Western blot analysis was performed as previously described (Miranda et al., 2010 (link)). We used the anti-TgVP1 polyclonal antibody at a dilution of 1:500 and secondary goat anti-Guinea pig antibody conjugated with HRP at 1:5,000. Mouse anti-α-tubulin at a dilution of 1:1,000 was used as a loading control.
Western blot analysis was performed as previously described (Miranda et al., 2010 (link)). We used the anti-TgVP1 polyclonal antibody at a dilution of 1:500 and secondary goat anti-Guinea pig antibody conjugated with HRP at 1:5,000. Mouse anti-α-tubulin at a dilution of 1:1,000 was used as a loading control.
