Clara cooled charge coupled device camera

Cells were extracted with MTSB (100 mM Pipes, pH 6.8, 5 mM MgCl₂, 1 mM EGTA, 30% glycerol, and 0.5% Triton X-100) for 2 min and fixed in 3.5% paraformaldehyde in PBS. Primary antibodies were diluted in TBS-BSA (10 mM Tris, pH 7.5, 150 mM NaCl, 10 mg/ml BSA, 0.1% NaN₃, and 0.1% Triton X-100) and incubated 1–24 h at 4°C. Secondary antibodies (goat anti–rabbit Alexa Fluor 488 or goat anti–mouse Alexa Fluor 568; Thermo Fisher Scientific) were diluted 1:1,000 in TBS-BSA and 1 µg/ml Hoechst 33258 (Sigma-Aldrich) and then incubated 1 h at room temperature. Coverslips were mounted on slides using ProLong Diamond (Molecular Probes). Images were acquired with a Clara cooled charge-coupled device camera (Andor Technology) mounted on an Axioplan2 microscope (ZEISS) with an Apochromat 100× 1.4 NA oil immersion objective. Exposure times were determined for each protein under WT conditions, and then the same time was applied to any mutants and siRNA-treated cells. Image channels were merged, contrast-adjusted (histogram stretching according to Cromey [2010] (link)), and assembled into figure panels in Photoshop (CS6 v13; Adobe).

Intracellular parasites were imaged ∼18 h after invasion into confluent HFF monolayers grown on MatTek dishes. Growth media was replaced with Fluorobrite DMEM (Life Technologies) containing 1% FBS and 1× antimycotic/antibiotic prewarmed to 37°C. Images were acquired using a Nikon Ti-E inverted microscope with a 100×, 1.49 NA objective in an environmental chamber heated to 37°C equipped with an Spectra-X light engine (Lumencore, Beaverton, OR) and a Clara cooled charge-coupled device camera (Andor, South Windsor, CT). Images were acquired at 100 ms for 1 min. Dense granule motions were tracked using the ImageJ, version 1.43, plug-in, SpotTracker2D (National Institutes of Health), allowing us to determine the granules’ positions at subpixel resolution in every frame of the movie so that change point and MSD analysis could be performed (see following section). Out-of-focus granules and granules that could not be spatially resolved from one another were eliminated from analysis. When necessary, the ImageJ plug-in “template matching” was used to compensate for drift of the PV inside the cell.