Alexa fluor 647 and alexa fluor 488 secondary antibodies

For immunofluorescence assays, cells were fixed with 4% paraformaldehyde for 15 min. The cells were then permeabilized and blocked for 60 min with 0.1 to 0.2% (vol/vol) Triton X-100 in phosphate-buffered saline (PBS) (pH 7.4), typically with 3% (wt/vol) goat serum. For T. gondii intracellular growth and survival assays, cells were incubated with mouse polyclonal anti-SAG1 (DG52) (67 (link)) at 1:10,000 overnight, followed by incubation with Alexa Fluor 647 secondary antibodies (Molecular Probes) for 1 h. For GBP5 localization assays, cells were incubated with anti-SAG1 (DG52) at 1:10,000 and anti-GBP5 (Cell Signaling Technologies, no. 67798) at 1:500 overnight followed by incubation with Alexa Fluor 647 and Alexa Fluor 488 secondary antibodies (Molecular Probes) for 1 h. For ROP16 localization assays, cells were incubated with anti-SAG1 (DG52) at 1:10,000 and anti-DYKDDDDK (Cell Signaling Technologies, no. 14793) at 1:500 overnight followed by incubation with Alexa Fluor 647 and Alexa Fluor 488 secondary antibodies (Molecular Probes) for 1 h. Coverslips were mounted on slides using Fluoromount G (ThermoFisher, 00-4958-02).