For immunofluorescence analysis, HFF cells were grown on coverslips and inoculated with T. gondii parasites in the absence and presence of ATc (at 1 mg/ml) for 48–50 h. To investigate the distribution of actin in extracellular parasites, growth in ATc was extended to 68–72 h, and freshly released tachyzoites were harvested. Cells were fixed with 4% (wt/vol) paraformaldehyde and 0.0075% glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA) in phosphate-buffered saline (PBS) for 10 min before permeabilization with 1% (vol/vol) Triton X-100 in PBS for 10 min, followed by blocking in 3% (wt/vol) bovine serum albumin (Sigma-Aldrich, St. Louis, MO) in PBS for 1 h. Primary antibodies were diluted in blocking solution and used in the following concentrations: rabbit anti-HA clone C29F4 (Cell Signaling Technology, Danvers, MA), 1:4000; mouse anti-cMyc clone 9E10 (Sigma-Aldrich), 1:2000; and rabbit anti-Act239-253 (Angrisano et al., 2012 (link)), 1:4000. Secondary antibodies used were Alexa Fluor 488 goat anti-mouse and Alexa Fluor 594 goat anti-rabbit (Life Technologies) in 1:4000 dilutions.
Fluorescence microscopy was performed using a DeltaVision Microscopy System (Applied Precision, Seattle, WA) and an Olympus UPlanSApo 60×/numerical aperture (NA) 1.4 or 100×/1.4 NA oil objective.
Fluorescence microscopy was performed using a DeltaVision Microscopy System (Applied Precision, Seattle, WA) and an Olympus UPlanSApo 60×/numerical aperture (NA) 1.4 or 100×/1.4 NA oil objective.
