Image pro plus 2

HFFs were grown to confluency in 24-well plates. For staining with MitoTracker (Invitrogen), medium was replaced with prewarmed DMEM containing MitoTracker at a concentration of 50 nM. After 30 min of incubation at 37°C, cells were washed and then infected with parasites in prewarmed cDMEM. At 4 hpi, cells were washed in PBS and fixed in prewarmed cDMEM containing 3.7% formaldehyde for 15 min. Otherwise, parasites were allowed to invade confluent HFF monolayers on coverslips for 6 and 12 h, fixed in 4% formaldehyde, permeabilized with 0.1% Triton-X100 for 20 min, and blocked in PBS supplemented with 3% BSA. Coverslips were then incubated with 3F10 (anti-HA) antibody (Roche, Palo Alto, CA), antibodies specific for TOM20 (SCBT, Santa Cruz, CA), or mouse polyclonal sera to MAF1 for 1–3 h at room temperature (RT). Fluorescent secondary antibodies (Invitrogen/Molecular Probes, Carlsbad, CA) were used for antigen visualization. Coverslips were mounted in VectaShield (Vector). Phase and fluorescence images were captured with a Hamamatsu Orca100 CCD on an Olympus BX60 (100×) and FV1000 Olympus confocal scope (DIC) and processed using Image-Pro Plus 2.0 (MediaCybernetics).