Axio observer z1 imaging system

HCC1937 cells and MEFs were fixed in methanol at −20^oC for 3 min and permeabilized in PBS-T (PBS with 0.1% Triton X-100) for 5 min at room temperature. Fixed cells were blocked with antibody dilution buffer (2% BSA, 0.1% Triton X-100 in TBS) for 1 hr at room temperature, and incubated in primary antibody at 4^oC overnight. A rabbit anti-Oct1 antibody was used (1:500; Santa Cruz Biotechnology sc-232X). Cells were probed with a secondary goat anti-rabbit antibody conjugated to Alexa488 (Invitrogen #A11008). Images were collected using a Zeiss Axio Observer Z1 imaging system.

Full-length ROP21 cDNA was amplified from TgME49 SMART cDNA and cloned into linear pTub-YFPYFP-CAT (Gubbels et al., 2003 ) (digested with BglII and AvrII to remove the first YFP copy) using NEBuilder HIFI Assembly mix (NEB). The validated plasmid was electroporated into RH and RHΔku80Δasp5 (Curt-Varesano et al., 2015 ) using 10 μg per transfection, and after 24 h chloramphenicol selection was applied at 20 μM for wild type RH and 10 μM for the Δasp5 background. After establishment of stable pools, parasites were cloned by limiting dilution. Expression of the protein was confirmed by western blotting using rabbit anti-GFP polyclonal serum (Molecular Probes) and mouse anti-SAG1 DG52 (Burg et al., 1988 (link)) as a loading control. For live cell imaging, parasites were seeded into HFFs growing in glass-bottomed culture dishes (MatTek, Ashland, MA) and allowed to replicate for 24-30 h. Infected cell monolayers were then imaged using a Zeiss AxioObserver Z1 imaging system and Zen Software (Drewry et al., 2015) equipped with a Cell Observer SD spinning disc confocal system with 488nm (50mW) and 561 nM (50mW) lasers (Zeiss) and an Evolve 512 EMCCD Camera (Photometrics).